miscript Plant qpcr System Handbook

Transcription

1 June 2014 miscript Plant qpcr System Handbook miscript Plant RT Kit miscript SYBR Green PCR Kit miscript mirna PCR Arrays miscript Primer Assays For SYBR Green real-time PCR expression profiling and quantification of plant micrornas and other 3 modified small RNAs Sample & Assay Technologies

2 QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies, enabling the isolation and detection of contents of any biological sample. Our advanced, high-quality products and services ensure success from sample to result. QIAGEN sets standards in: Purification of DNA, RNA, and proteins Nucleic acid and protein assays microrna research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs. For more information, visit

3 Contents Kit Contents 4 Storage 7 Intended Use 8 Safety Information 8 Quality Control 8 Introduction 9 Principle and procedure 11 Template RNA requirements 16 Equipment and Reagents to Be Supplied by User 17 Protocols Ligation-mediated reverse-transcription 18 Real-Time PCR using plant miscript mirna PCR Arrays 22 Real-Time PCR using plant miscript Primer Assays 28 Data analysis using the C T method of relative quantification 31 Troubleshooting Guide 35 References 39 Appendix A: Isolation of total RNA, including small RNAs, from plant tissues 39 Appendix B: Real-Time PCR data output and dissociation curve analysis 42 Appendix C: General remarks on handling RNA 45 Appendix D: Preparation, quantification, and storage of RNA 47 Ordering Information 51 miscript Plant qpcr System Handbook 06/2014 3

4 Kit Contents miscript Plant RT Kit (12) (50) (96) Catalog no Number of preps x miscript Ligation Buffer 24 µl 100 µl 2 x 100 µl 10x miscript Plant Adaptor 24 µl 100 µl 2 x 100 µl 2x miscript Ligation Activator 120 µl 500 µl 2 x 500 µl miscript Plant RNA Ligase 12 µl 50 µl 2 x 50 µl 5x miscript Plant RT Buffer 48 µl 200 µl 2 x 200 µl 10x miscript Plant RT Nucleics 24 µl 100 µl 2 x 100 µl miscript Plant Reverse Transcriptase 12 µl 50 µl 2 x 50 µl RNase-Free Water 1.0 ml 1.0 ml 2 x 1.0 ml Quick-Start Protocol miscript Plant qpcr System Handbook 06/2014

5 miscript SYBR Green PCR Kit (200)* (1000) (2000) Catalog no Number of preps x QuantiTect SYBR Green PCR Master Mix, containing: HotStarTaq DNA Polymerase QuantiTect SYBR Green PCR Buffer dntp mix, including dutp SYBR Green I ROX passive reference dye 5 mm MgCl 2 3 x 1.7 ml 25 ml 2 x 25 ml 10x miscript Universal Primer 1 ml 5 x 1 ml 10 x 1 ml RNase-Free Water 2 x 2 ml 20 ml 2 x 20 ml Quick-Start Protocol * The miscript SYBR Green PCR Kit (200) provides sufficient reagents for 3 x 96-well arrays (975 µl residual), 2 x 384-well (4 x 96, 4 sample) arrays (700 µl residual), 2 x 384-well (HC, 1 sample) arrays (1000 µl residual), or 4 Rotor-Disc 100 arrays (700 µl residual). The miscript SYBR Green PCR Kit (1000) provides sufficient reagents for 18 x 96-well arrays (250 µl residual), 11 x 384-well (4 x 96, 4 sample) arrays (800 µl residual), 12 x 384-well (HC, 1 sample) arrays (400 µl residual), or 22 Rotor-Disc 100 arrays (800 µl residual). The miscript SYBR Green PCR Kit (2000) provides sufficient reagents for 36 x 96-well arrays (500 µl residual), 22 x 384-well (4 x 96, 4 sample) arrays (1600 µl residual), 24 x 384-well (HC, 1 sample) arrays (800 µl residual), or 45 Rotor-Disc 100 arrays (500 µl residual). miscript Plant qpcr System Handbook 06/2014 5

6 miscript mirna PCR Arrays Catalog no. Format A C D E F G R 96-well plate containing dried assays Varies Varies Varies Varies Varies 384-well plate containing dried assays Varies Varies Rotor-Disc 100 containing dried assays Optical Thin-Wall 8-Cap Strips (12 per plate) Optical Adhesive Film (1 per plate) Rotor-Disc Heat Sealing Film (1 per Rotor-Disc) Varies Varies Varies Varies Varies Varies Varies Varies miscript Primer Assay (100) Catalog no. Varies* Number of 50 µl reactions x miscript Primer Assay (contains one mirna-specific primer) 1 vial * Visit to search for and order assays. The 10x miscript Primer Assay is supplied lyophilized and must be reconstituted according to the instructions in Storage (Page 7). 6 miscript Plant qpcr System Handbook 06/2014

7 Cyclers for use with array formats Array Format Suitable real-time cyclers A Agilent /Stratagene models Mx3005P, Mx3000P ; Applied Biosystems models 5700, 7000, 7300, 7500, 7700, 7900HT, ViiA 7 (96-well block); Bio- Rad models icycler, iq 5, MyiQ, MyiQ2; Bio- Rad/MJ Research Chromo4 ; Eppendorf Mastercycler ep realplex models 2, 2S, 4, 4S; Takara TP-800 Plate 96-well C D E Applied Biosystems models 7500 (Fast block), 7900HT (Fast block), StepOnePlus, ViiA 7 (Fast block) Bio-Rad CFX96 ; Bio-Rad/MJ Research models DNA Engine Opticon, DNA Engine Opticon 2; Stratagene Mx4000 Applied Biosystems models 7900HT (384-well block), ViiA 7 (384-well block); Bio-Rad CFX well 96-well 384-well F Roche LightCycler 480 (96-well block) 96-well G Roche LightCycler 480 (384-well block) 384-well R Rotor-Gene Q; Rotor-Gene 6000; other Rotor-Gene cyclers Rotor-Disc 100 Storage The miscript Plant RT Kit and miscript SYBR Green PCR Kit are shipped on dry ice. The kits, including all reagents and buffers, should be stored immediately upon receipt at 20ºC in a constant-temperature freezer. miscript mirna PCR Arrays are shipped at ambient temperature, on ice, or on dry ice depending on the destination and accompanying products. Upon receipt, store at 20 C. If stored under these conditions, miscript mirna PCR Arrays are stable for 6 months after receipt. miscript Primer Assays are shipped lyophilized at room temperature. Store them at 20ºC, either lyophilized or reconstituted (see next paragraphs). Avoid repeated freeze thaw cycles. When stored under these conditions and handled correctly, the product can be kept for at least 18 months from date of receipt miscript Plant qpcr System Handbook 06/2014 7

8 without reduction in performance. To reconstitute 10x miscript Primer Assay, briefly centrifuge the vial, add 550 µl RNase-free water, and mix by vortexing the vial 4 6 times. This will provide sufficient primer for 200 x 25 µl reactions. QIAGEN recommends freezing the reconstituted primers in aliquots in order to avoid repeated freezing and thawing. Intended Use The miscript Plant RT Kit, miscript SYBR Green PCR Kit, miscript mirna PCR Arrays, and miscript Primer Assays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease. All due care and attention should be exercised in the handling of the products. We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments, or to other applicable guidelines. Safety Information When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate safety data sheets (SDSs). These are available online in convenient and compact PDF format at where you can find, view, and print the SDS for each QIAGEN kit and kit component. Quality Control In accordance with QIAGEN s ISO-certified Quality Management System, each lot of miscript Plant RT Kit, miscript SYBR Green PCR Kit, miscript mirna PCR Array, and miscript Primer Assay is tested against predetermined specifications to ensure consistent product quality. 8 miscript Plant qpcr System Handbook 06/2014

9 Introduction The miscript Plant qpcr System consists of the miscript Plant RT Kit, miscript SYBR Green PCR Kit, miscript mirna PCR Arrays, miscript Primer Assays, and the GeneGlobe Data Analysis Center. The miscript Plant qpcr System allows sensitive and specific detection and quantification of plant mature microrna (mirna). The miscript Plant qpcr System uses total RNA that contains mirna as the starting material for cdna synthesis, and separate enrichment of small RNA is not needed. A single cdna preparation can be used with either miscript mirna PCR Arrays or miscript Primer Assays for plant mature mirna expression profiling and quantification. miscript Plant RT Kit Small RNAs possessing a 2 -O-Me modification on their 3 terminal base, such as plant mature mirnas and piwi-interacting RNAs (pirnas), are refractory to polyadenylation. As a result, they cannot be efficiently reverse transcribed using a polyadenylation-based cdna synthesis approach, which is the underlying mechanism of various mirna reverse-transcription kits including the miscript II RT Kit. In response to this, the specialized miscript Plant RT Kit has been developed. With the miscript Plant RT Kit, an adaptor is first ligated to the 3 terminus of plant mirnas. Unlike polyadenylation, this ligation reaction is not inhibited by the presence of a 2 -O-Me modification on the 3 terminal base of plant mirnas. Following the ligation, reverse-transcription is performed. The miscript Plant RT Kit represents an unbiased, universal cdna synthesis kit for small RNAs with 3 -end, 2 -O-Me modifications. miscript mirna PCR Arrays miscript mirna PCR Arrays are mature mirna-specific forward primers (miscript Primer Assays) that have been arrayed in pre-formatted panels. Offered for several plant species, miscript PCR arrays are available in ready-touse 384-well plate, 96-well plate, and 100-well Rotor-Disc formats. In addition, miscript PCR arrays provide guaranteed high performance and are fully customizable for user-selected content from the QIAGEN catalog of verified miscript Primer Assays. Each assay in a miscript PCR array has been benchverified to ensure sensitive and specific detection of mature mirna by real-time PCR. The complimentary, web-based GeneGlobe Data Analysis Center simplifies the analysis of real-time PCR data. Once threshold cycle (CT) data has been uploaded, the tool automatically performs all fold-change calculations using the ΔΔC T method of relative quantification, and presents the results in several formats. miscript mirna PCR Arrays are at the forefront of real-time PCR-based mature mirna profiling tools. miscript Plant qpcr System Handbook 06/2014 9

10 dfsdf Figure 1. miscript Plant qpcr System workflow. 10 miscript Plant qpcr System Handbook 06/2014

11 Principle and procedure Mature mirnas are naturally occurring, 22-nucleotide, noncoding RNAs that mediate posttranscriptional gene regulation. Plant mirnas and pirnas possess a 2 -O-Me residue on their 3 terminal base. Due to this, ligation-mediated reverse-transcription is required for the universal cdna synthesis of plant mirnas and pirnas. Ligation-mediated reverse-transcription The miscript Plant RT Kit includes all of the reagents required to perform universal, ligation-mediated reverse-transcription of plant mature mirnas, including 10x miscript Ligation Buffer, 10x miscript Plant Adaptor, 2x miscript Ligation Activator, miscript Plant RNA Ligase, 10x miscript Plant RT Nucleics, 5x miscript Plant RT Buffer, and miscript Plant Reverse Transcriptase. When ligation-mediated reverse-transcription is performed, small RNAs are selectively converted into cdna. In the first step of the reaction, an adaptor is ligated to the mature mirnas. In the second step of the reaction, the ligated mirnas are reverse-transcribed into cdna (Figure 2). 10x miscript Plant Adaptor contains an optimized pre-adenylated adaptor and a synthetic RNA control (miscript Plant Ligation Control [miplc]) that is used to assess ligation-mediated reverse-transcription performance. 10x miscript Plant RT Nucleics contains dntps, RT primers, and an internal synthetic RNA-DNA hybrid control (miscript Plant Reverse-Transcription Control [miprc]) that is used to test reverse-transcription performance. The unique, patent-pending formulation of 5x miscript Plant RT Buffer facilitates the selective conversion of mature mirnas into cdna, enabling mirna quantification with either miscript mirna PCR Arrays or miscript Primer Assays. Real-time PCR mirna expression profiling cdna prepared using the miscript Plant RT Kit serves as the template for realtime PCR analysis using a plant miscript mirna PCR Array (which contains mirna-specific miscript Primer Assays) and the miscript SYBR Green Kit, which contains the miscript Universal Primer (reverse primer) and QuantiTect SYBR Green PCR Master Mix. To profile mature mirna expression, a premix of cdna, miscript Universal Primer, QuantiTect SYBR Green PCR Master Mix, and RNase-free water is added to a miscript mirna PCR Array. Real-time PCR mirna expression quantification cdna prepared using the miscript Plant RT Kit serves as the template for realtime PCR analysis using a plant miscript Primer Assay and the miscript SYBR Green Kit. To quantify mature mirna expression, a mix of cdna, miscript miscript Plant qpcr System Handbook 06/

12 Primer Assay, miscript Universal Primer, QuantiTect SYBR Green PCR Master Mix, and RNase-free water is prepared and added to PCR tubes, Rotor-Discs, or plates. Figure 2. Conversion of mature mirnas into cdna and subsequent detection. In a miscript Plant RT Kit reaction, mature mirnas are first ligated to an adaptor. In a second enzymatic reaction, the ligated mirnas are reverse-transcribed into cdna. The cdna is then used for real-time PCR expression analysis with either miscript mirna PCR Arrays or miscript Primer Assays. miscript mirna PCR Array plate layout miscript mirna PCR Arrays are available in 96-well, 384-well, and Rotor-Disc 100 formats (Figures 3 6). Each array contains several control assays. The purpose of each control is described on page miscript Plant qpcr System Handbook 06/2014

13 Figure well miscript mirna PCR Array layout for plate formats A, C, D, and F. Wells A1 to G12 (1 84) each contain an individual miscript Primer Assay. Wells H1 to H6 each contain an assay for a different species-specific snorna or snrna that can be used as a normalization control for the array data (SN1, SN2, SN3, SN4, SN5, SN6). Wells H7 and H8 contain replicate ligation controls (miplc). Wells H9 and H10 contain replicate reversetranscription controls (miprc). Wells H11 and H12 contain replicate positive PCR controls (PPC). Figure well (4 x 96, 4 sample) miscript mirna PCR Array layout for plate formats E and G. Focused miscript mirna PCR Arrays in formats E and G include 4 replicates of the same assays as provided in the 96-well format shown in Figure 3. miscript Plant qpcr System Handbook 06/

14 Figure well (HC, 1 sample) miscript mirna PCR Array layout for plate formats E and G. High-content miscript mirna HC PCR Arrays in formats E and G include 384 unique assays in the 384-well plate format. Wells A1 to P12 (1 372) each contain an individual miscript Primer Assay. Wells H13 to H18 each contain an assay for a different species- specific snorna or snrna that can be used as a normalization control for the array data (SN1, SN2, SN3, SN4, SN5, SN6). Wells H19 and H20 contain replicate ligation controls (miplc). Wells H21 and H22 contain replicate reverse-transcription controls (miprc). Wells H23 and H24 contain replicate positive PCR controls (PPC). Figure 6. miscript mirna PCR Array layout for Rotor-Disc format R. Wells 1 84 each contain an individual miscript Primer Assay. Wells each contain an assay for a different species-specific snorna or snrna that can be used as a normalization control for the array data (SN1, SN2, SN3, SN4, SN5, SN6). Wells 91 and 92 contain replicate ligation controls (miplc). Wells 93 and 94 contain replicate reverse-transcription controls (miprc). Wells 95 and 96 contain replicate positive PCR controls (PPC). Wells are empty. 14 miscript Plant qpcr System Handbook 06/2014

15 Controls in miscript mirna PCR Arrays The final 12 wells of each miscript mirna PCR Array contain a series of controls (Table 1). Table 1. Controls in miscript PCR Arrays Control 6 species-specific snorna or snrna miscript Primer Assays miscript Plant Ligation Control (miplc) miscript Plant Reverse-Transcription Control (miprc) Positive PCR control (PPC) Purpose Data normalization using the C T method of relative quantification Assessment of ligation-mediated reverse-transcription performance Assessment of reverse-transcription performance Assessment of PCR performance For accurate and reproducible results in mirna quantification by real-time PCR, it is necessary to normalize the amount of target mirna by using a suitable endogenous reference RNA. This method is known as relative quantification. Normalization corrects for factors that could otherwise lead to inaccurate quantification. These factors include variation in quantity of input RNA, possible RNA degradation or presence of inhibitors in the RNA samples, and differences in sample handling. Normalization also allows results from different experiments and samples to be compared directly. For each plant species, miscript PCR Controls have been designed to quantify a panel of snornas or snrnas. These small RNAs have been verified to have robust, stable expression in their respective species. In addition, all controls have amplification efficiencies close to 100%. As a result, miscript PCR Controls are ideal normalization controls for relative quantification using the miscript Plant qpcr System. Besides selecting up to 6 snorna/snrna controls, other potential normalization options include selecting an invariant mirna or calculating a plate mean of expressed mirnas. The miscript Plant Ligation Control (miplc) is an assay that assesses combined ligation/reverse-transcription performance by detecting template synthesized from the kit s built-in control RNA. This control monitors for any variables that may inhibit the ligation-mediated reverse-transcription reaction. The miscript Plant Reverse-Transcription Control (miprc) is an assay that assesses reverse-transcription performance by detecting template synthesized miscript Plant qpcr System Handbook 06/

16 from the kit s built-in control DNA-RNA hybrid. This control monitors for any variables that may exclusively inhibit the reverse-transcription reaction. The positive PCR control (PPC) wells contain a predispensed artificial DNA sequence and the assay that detects it. This control monitors for any variables that may inhibit the PCR reaction. Data analysis Free data analysis software for miscript mirna PCR Arrays and miscript Primer Assays is available through the GeneGlobe Data Analysis Center. Data analysis and interpretation of the control assay is automatically performed using the C T method of relative quantification and interpretation of the control assays (for more details, see page 31). Results are presented in a tabular format, a scatter plot, a three-dimensional profile, and a volcano plot (when replicates are included). Template RNA requirements Total RNA containing mirna is the required starting material for plant miscript mirna PCR Arrays and miscript Primer Assays. It is not necessary to enrich for small RNA. The mirneasy Supplementary Protocol found in Appendix A should be followed for purification of total RNA including mirna from plant tissues. A generic protocol for RNA extraction from plants is difficult to suggest, as plants differ widely in their physical makeup and can contain large amounts of problematic material which can interfere with downstream analysis. The protocol described in Appendix A should be sufficient for simple green leaves, soft roots, and other plant parts. We recommend a literature search for publications detailing more specialized isolation procedures for your specific sample type, or call QIAGEN technical support for assistance. 16 miscript Plant qpcr System Handbook 06/2014

17 Equipment and Reagents to Be Supplied by User When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier. For ligation-mediated reverse-transcription Thin-walled, DNase-free, RNase-free PCR tubes (for 20 µl reactions) Ice Thermal cycler or chilling/heating block Microcentrifuge For quantitative, real-time PCR Real-time PCR cycler; the table on page 7 shows the appropriate real-time cycler for each array format Multichannel pipettor Nuclease-free pipet tips and tubes miscript Plant qpcr System Handbook 06/

18 Protocol: Ligation-mediated reverse-transcription Important points before starting Total RNA, containing mirna, should be used as starting material. For a protocol detailing total RNA purification from plant tissues, see Appendix A. This protocol is for use with a maximum of 500 ng to a minimum of 50 pg of RNA. Recommended starting amounts are shown in Table 2. If working with RNA for the first time, read Appendix C (page 45). Set up ligation reactions at room temperature. Set up reverse-transcription reactions on ice. Transfer exactly 4 µl of the ligated product into the subsequent reversetranscription reaction. Do not vortex template RNA or any component of the miscript Plant RT Kit. Table 2. Recommended RNA starting amounts PCR application Assay Recommended RNA input* Mature mirna quantification miscript Primer Assays ng per RNA sample Mature mirna expression profiling Mature mirna expression profiling 96-well and 384- well (4 x 96, 4 sample) miscript mirna PCR Arrays 384-well (HC, 1 sample) or 4 x 96 miscript mirna PCR Arrays ng per RNA sample ng per intended 384-well (HC, 1 sample) plate or 4 x 96-well plates / Rotor-Discs * If the RNA sample is not limiting, use the larger recommendation. Procedure 1. Prepare reagents required for ligation. Thaw template RNA on ice. Thaw RNase-Free Water, 10x miscript Ligation Buffer, 10x miscript Plant Adaptor, and 2x miscript Ligation Activator at room temperature (15 25ºC). Mix each solution by flicking the tubes. Centrifuge the tubes briefly to collect residual 18 miscript Plant qpcr System Handbook 06/2014

19 liquid from the sides of the tubes and keep at room temperature. Note: miscript Plant RNA Ligase should be removed from the 20ºC freezer just before preparation of the master mix, gently mixed, and placed on ice. The miscript Plant RNA Ligase should be returned to the freezer immediately after use. 2. Prepare the ligation reaction at room temperature (15 25ºC) according to Table 3. Briefly centrifuge, mix by pipetting up and down 12 times, and briefly centrifuge again. Note: If setting up more than one reaction, prepare a volume of reaction mix 10% greater than that required for the total number of reactions to be performed. Note: 2x miscript Ligation Activator is very viscous. Add 2x miscript Ligation Activator individually at Step 4, and do not include 2x miscript Ligation Activator when preparing a mix for multiple reactions. Table 3. Ligation reaction components Component Volume/reaction 10x miscript Ligation Buffer 2 µl 10x miscript Plant Adaptor 2 µl RNase-Free Water Variable miscript Plant RNA Ligase 1 µl RNA Template (added in step 3) 2x miscript Ligation Activator (added in step 4) Variable 10 µl Total volume 20 µl 3. Add template RNA to each tube containing ligation mix. 4. Slowly add 2x miscript Ligation Activator to each tube containing ligation mix and RNA. Briefly centrifuge, mix by pipetting up and down 12 times, and briefly centrifuge again. Note: Pipet slowly to mix. 2x miscript Ligation Activator is very viscous. 5. Incubate for 1 hr at 16ºC. 6. Incubate for 20 min at 65ºC to inactivate the miscript Plant RNA Ligase and hold at 4ºC. 7. Prepare reagents required for reverse-transcription. Thaw 5x miscript Plant RT Buffer and 10x miscript Plant RT Nucleics at room temperature (15 miscript Plant qpcr System Handbook 06/

20 25ºC). Mix each solution by flicking the tubes. Centrifuge the tubes briefly to collect residual liquid from the sides of the tubes and then store on ice. 8. Immediately prior to setting up the RT reactions, allow the completed ligation reactions to equilibrate to room temperature for 2 min. Briefly centrifuge, mix by pipetting up and down twelve times, and briefly centrifuge. 9. Prepare the reverse-transcription reaction on ice according to Table 4. Briefly centrifuge, mix by pipetting up and down eight times, briefly centrifuge, and store on ice. The reverse-transcription master mix contains all components required for first-strand cdna synthesis except the ligation product of the upstream ligation reaction. Note: miscript Plant Reverse Transcriptase should be removed from the 20ºC freezer just before preparation of the master mix, gently mixed by tapping, and placed on ice. miscript Plant Reverse Transcriptase should be returned to the freezer immediately after use. Note: If setting up more than one reaction, prepare a volume of reaction mix 10% greater than that required for the total number of reactions to be performed. Table 4. Reverse-transcription reaction components Component Volume/reaction 5x miscript Plant RT Buffer 4 µl 10x miscript Plant RT Nucleics 2 µl RNase-Free Water 9 µl miscript Plant Reverse Transcriptase 1 µl Ligation Product (added in step 10) 4 µl Total volume 20 µl 20 miscript Plant qpcr System Handbook 06/2014

21 10. Add 4 µl of the ligation product from Step 8 to each tube containing reverse-transcription mix. Briefly centrifuge, mix by pipetting up and down 8 times, briefly centrifuge, and then store on ice. Note: Exactly 4 µl of the ligation product must be added to the reversetranscription reaction. 11. Incubate for 2 hr at 37ºC. 12. Incubate for 5 min at 95ºC to inactivate the miscript Plant Reverse Transcriptase and hold at 4ºC. 13. Dilute the cdna in RNase-free water according to Table 5, and proceed with real-time PCR immediately. If you wish to store the reverse-transcription reactions prior to real-time PCR, transfer the undiluted cdna to a 20ºC freezer, or dispense the diluted cdna into 110 µl aliquots and transfer them to a 20ºC freezer. Table 5. cdna dilution prior to PCR PCR application Assay Reaction dilution Mature mirna quantification miscript Primer Assays Add 200 µl RNase-free water to each 20 µl reverse-transcription reaction Mature mirna expression profiling Mature mirna expression profiling 96-well and 384- well (4 x 96, 4 sample) miscript mirna PCR Arrays 384-well (HC, 1 sample) or 4 x 96 miscript mirna PCR Arrays Add 200 µl RNase-free water to each 20 µl reverse-transcription reaction Dilution depends on the number of intended 384-well (HC, 1 sample) or 4 x 96 plates/rotor-discs: For 1 x 384-well (HC, 1 sample) plate or 4 x 96 plates/rotor-discs, add 90 µl RNase-free water to the 20 µl reverse-transcription reaction For 2 x 384-well (HC, 1 sample) plate or 8 x 96 plates/rotor-discs, add 200 µl RNase-free water to the 20 µl reverse-transcription reaction miscript Plant qpcr System Handbook 06/

22 Protocol: Real-Time PCR using plant miscript mirna PCR Arrays cdna prepared using the miscript Plant RT Kit is the appropriate starting material for this protocol. This protocol enables real-time PCR profiling of mature mirna using miscript mirna PCR Arrays in combination with the miscript SYBR Green PCR Kit, which contains the miscript Universal Primer (reverse primer) and QuantiTect SYBR Green Master Mix. Important points before starting The PCR must start with an initial incubation step of 15 minutes at 95ºC to activate HotStarTaq DNA Polymerase (included in 2x QuantiTect SYBR Green PCR Master Mix). Only cdna template prepared using the miscript Plant RT Kit should be used with this protocol. Ensure that the 20 µl cdna synthesis reaction has been diluted appropriately. See Table 5 for recommendations. Do not vortex template cdna or any of the components of the miscript SYBR Green PCR Kit. The miscript SYBR Green PCR Kit (200) provides sufficient reagents for 3 x 96-well arrays (975 µl residual), 2 x 384-well (4 x 96, 4 sample) arrays (700 µl residual), 2 x 384-well (HC, 1 sample) arrays (1000 µl residual), or 4 Rotor-Disc 100 arrays (700 µl residual). The miscript SYBR Green PCR Kit (1000) provides sufficient reagents for 18 x 96-well arrays (250 µl residual), 11 x 384-well (4 x 96, 4 sample) arrays (800 µl residual), 12 x 384-well (HC, 1 sample) arrays (400 µl residual), or 22 Rotor-Disc 100 arrays (800 µl residual). The miscript SYBR Green PCR Kit (2000) provides sufficient reagents for 36 x 96-well arrays (500 µl residual), 22 x 384-well (4 x 96, 4 sample) arrays (1600 µl residual), 24 x 384-well (HC, 1 sample) arrays (800 µl residual), or 45 Rotor-Disc 100 arrays (500 µl residual). If using the icyler iq, iq5, or MyiQ, well factors must be collected at the beginning of each experiment. Well factors are used to compensate for any system or pipetting non-uniformity. For details, refer to the user manual supplied with the instrument or Technical Information: Using QuantiTect SYBR Green Kits on Bio-Rad cyclers available at 22 miscript Plant qpcr System Handbook 06/2014

23 Procedure 1. Thaw 2x QuantiTect SYBR Green PCR Master Mix, 10x miscript Universal Primer, template cdna, and RNase-free water at room temperature (15 25ºC). Mix each solution by flicking the tubes. Centrifuge the tubes briefly to collect residual liquid from the sides of the tubes and then store on ice. 2. Prepare a reaction mix according to Table 6 or Table 7, depending on your application. Mix thoroughly but gently. Due to the hot start, it is not necessary to keep samples on ice during reaction setup or while programming the real-time cycler. Table 6. Reaction mix for Focused miscript mirna PCR Arrays* Array format: Component 384-well (4 x 96, 4 sample) Formats E, G 96-well Formats A, C, D, F Rotor-Disc 100 Format R 2x QuantiTect SYBR Green PCR Master Mix 10x miscript Universal Primer 550 µl 1375 µl 1100 µl 110 µl 275 µl 220 µl RNase-free water 340 µl 1000 µl 780 µl Template cdna 100 µl 100 µl 100 µl Total volume 1100 µl 2750 µl 2200 µl * These volumes provide a 10 µl per well reaction volume for a 384-well plate, 25 µl per well reaction volume for a 96-well plate, and 20 µl per well reaction volume for a 100-well Rotor- Disc. Volumes shown are sufficient for one cdna template. In total, 4 cdna templates can be analyzed on one 384-well (4 x 96, 4 sample) Focused miscript mirna PCR Array because assays are arrayed in quadruplicate (see Figure 4, page 13). No optimization of the Mg 2+ concentration is required. The final Mg 2+ concentration provided by 2x QuantiTect SYBR Green PCR Master Mix gives optimal results. miscript Plant qpcr System Handbook 06/

24 Table 7. Reaction mix for 384-well (HC, 1 sample) and 96-well/Rotor-Disc array sets* Array format: Component 384-well (HC, 1 sample) Formats E, G 96-well Formats A, C, D, F Rotor-Disc 100 Format R 2x QuantiTect SYBR Green PCR Master Mix 10x miscript Universal Primer RNase-free water 2050 µl 1375 µl 1100 µl 410 µl 275 µl 220 µl 1540 µl 1075 µl 855 µl Template cdna 100 µl 25 µl 25 µl Total volume 4100 µl 2750 µl 2200 µl * Volumes are for a single 384-well (HC, 1 sample) or 96-well plate/rotor-disc. Scale up volumes according to the number of plates/rotor-discs to be run. If a plate/rotor-disc is less than half full, scale down volumes accordingly. These volumes provide a 10 µl per well reaction volume for a 384-well plate, 25 µl per well reaction volume for a 96-well plate, and 20 µl per well reaction volume for a 100-well Rotor- Disc. No optimization of the Mg 2+ concentration is required. The final Mg 2+ concentration provided by 2x QuantiTect SYBR Green PCR Master Mix gives optimal results. 3. Carefully remove the miscript mirna PCR Array from its sealed bag. Optional for 96-well and 384-well array formats: If the reaction mix is in a tube, transfer to a loading reservoir, such as the RT 2 PCR Array Loading Reservoir (cat. no ). 4. Add reaction mix to each well of the miscript mirna PCR Array as follows. Note: For 384-well and 96-well array formats, a multichannel pipettor can be used to add reaction mix to the array. For the Rotor-Disc 100 format, a repeater pipettor or a QIAgility can be used to load the array. For 384-well miscript mirna PCR Array: add 10 µl per well. For 96-well miscript mirna PCR Array: add 25 µl per well. For Rotor-Disc miscript mirna PCR Array: add 20 µl per well. 24 miscript Plant qpcr System Handbook 06/2014

25 Loading 384-well (4 x 96, 4 sample) miscript mirna PCR Arrays Note: Each 384-well (4 x 96, 4 sample) miscript mirna PCR Array contains 4 replicates of 96 assays that can be used for analysis of 4 samples. The spacing between the tips of standard multichannel pipettors allows rows or columns to be skipped when adding each sample. Be sure to load each sample into the correct set of wells using a multichannel pipettor and the 384EZLoad Covers (provided). Use Figure 7 as a guide. Do not reuse 384EZLoad Covers. Place 384EZLoad Cover 1 (white) on the plate. Add 10 µl reaction mix for sample 1 to the open wells (odd number wells of rows A, C, E, G, I, K, M, and O). Remove and discard 384EZLoad Cover 1. Place 384EZLoad Cover 2 (yellow) on the plate. Add 10 µl reaction mix for sample 2 to the open wells (even number wells of rows A, C, E, G, I, K, M, and O). Remove and discard 384EZLoad Cover 2. Place 384EZLoad Cover 3 (black) on the plate. Add 10 µl reaction mix for sample 3 to the open wells (odd number wells of rows B, D, F, H, J, L, N, and P). Remove and discard 384EZLoad Cover 3. Place 384EZLoad Cover 4 (red) on the plate. Add 10 µl reaction mix for sample 4 to the open wells (even number wells of rows B, D, F, H, J, L, N, and P). Remove and discard 384EZLoad Cover 4. Cover 1 (white) for sample 1 Cover 2 (yellow) for sample 2 miscript Plant qpcr System Handbook 06/

26 Cover 3 (black) for sample 3 Cover 4 (red) for sample 4 Figure 7. Loading 384-well (4 x 96, 4 samples) miscript mirna PCR Arrays. Add 10 µl reaction mix for each of 4 samples into the staggered wells with the same number as indicated in the figure. 5. Carefully, tightly seal the miscript mirna PCR Array with Optical Thin-Wall 8-Cap Strips (Formats A and D), Optical Adhesive Film (Formats C, E, F, and G), or Rotor-Disc Heat-Sealing Film (Format R). 6. Centrifuge the PCR plate for 1 min at 1000 x g at room temperature (15 25 C) to remove bubbles. Note: This step is not necessary for reactions set up in Rotor-Discs. 7. Program the real-time cycler according to Table 8. Note: Perform dissociation curve analysis of the PCR product(s) to verify their specificity and identity. Dissociation curve analysis is an analysis step built into the software of real-time cyclers. Follow the instructions provided by the supplier. 26 miscript Plant qpcr System Handbook 06/2014

27 Table 8. Cycling conditions for real-time PCR Step Time Temperature Additional comments PCR initial activation step 3-step cycling:* 15 min 95ºC HotStarTaq DNA Polymerase is activated by this heating step. Denaturation 15 s 94ºC Annealing 30 s 55ºC Extension 30 s 70ºC Perform fluorescence data collection. Cycle number 40 cycles Cycle number depends on the amount of template cdna and abundance of the target. * For Eppendorf Mastercyler ep realplex models 2, 2S, 4, and 4S: for the Silver Thermoblock, adjust the ramp rate to 26%; for the Aluminum Thermoblock, adjust the ramp rate to 35%. If using a Roche LightCycler 480, adjust the ramp rate to 1.6 C/s. Due to software requirements, the fluorescence detection step must be at least 30 s with the ABI PRISM 7000 or 34 s with the Applied Biosystems 7300 and If using the Roche LightCycler 480, use 45 cycles. 8. Place the plate/rotor-disc in the real-time cycler and start the cycling program. 9. Once the real-time PCR run has been completed, continue to Protocol: Data analysis using the C T method of relative quantification. miscript Plant qpcr System Handbook 06/

28 Protocol: Real-Time PCR using plant miscript Primer Assays cdna prepared using the miscript Plant RT Kit is the appropriate starting material for this protocol. This protocol enables real-time PCR profiling of mature mirna using target-specific miscript Primer Assays in combination with the miscript SYBR Green PCR Kit, which contains the miscript Universal Primer (reverse primer) and QuantiTect SYBR Green Master Mix. Important points before starting The PCR must start with an initial incubation step of 15 minutes at 95ºC to activate HotStarTaq DNA Polymerase (included in 2x QuantiTect SYBR Green PCR Master Mix). Only cdna template prepared using the miscript Plant RT Kit should be used with this protocol. Ensure that the 20 µl cdna synthesis reaction has been diluted appropriately. See Table 5 for recommendations. Do not vortex template cdna or any of the components of the miscript SYBR Green PCR Kit. Things to do before starting If using the miscript Primer Assay for the first time, be sure to reconstitute it before use according to the instructions in Storage, page 7. (The miscript Universal Primer is provided as a ready-to-use 10x solution and does not need to be reconstituted). Procedure 1. Thaw 2x QuantiTect SYBR Green PCR Master Mix (if stored at 20ºC), 10x miscript Universal Primer, 10x miscript Primer Assay, template cdna, and RNase-free water. Mix each solution by flicking the tubes. Centrifuge the tubes briefly to collect residual liquid from the sides of the tubes and then store on ice. 2. Prepare a reaction mix according to Table 9 for either a 10 µl volume reaction (used for 384-well plates), a 25 µl volume reaction (used for 96- well plates), or a 20 µl volume reaction (used for Rotor-Disc 100). Reaction mix contains everything except the template cdna. Due to the hot start, it is 28 miscript Plant qpcr System Handbook 06/2014

29 not necessary to keep samples on ice during reaction setup or while programming the real-time cycler. Table 9. Reaction setup for real-time PCR Component 2x QuantiTect SYBR Green PCR Master Mix* 10x miscript Universal Primer 10x miscript Primer Assay RNase-free water Template cdna (added at step 3) Volume/reaction (384-well) Volume/reaction (96-well) Volume/reaction (Rotor-Disc 100) 5 µl 12.5 µl 10 µl 1 µl 2.5 µl 2 µl 1 µl 2.5 µl 2 µl 2 µl 6.5 µl 5 µl 1 µl 1 µl 1 µl Total volume 10 µl 25 µl 20 µl * No optimization of the Mg 2+ concentration is required. The final Mg 2+ concentration provided by 2x QuantiTect SYBR Green PCR Master Mix gives optimal results. 3. Gently, yet thoroughly, mix the reaction and dispense appropriate volumes into the plate wells containing template cdna. 4. Dispense template cdna into the individual wells of the PCR plate. 5. Seal the plates or Rotor-Discs with the appropriate films or caps. 6. Centrifuge plates for 1 min at 1000 x g to remove any bubbles. Note: This step is not necessary when using Rotor-Discs. 7. Program the real-time cycler according to Table 10. miscript Plant qpcr System Handbook 06/

30 Table 10. Cycling conditions for real-time PCR* Step Time Temperature Additional comments PCR Initial activation step 3-step cycling:* 15 min 95ºC HotStarTaq DNA Polymerase is activated by this heating step. Denaturation 15 s 94ºC Annealing 30 s 55ºC Extension 30 s 70ºC Perform fluorescence data collection. Cycle number 40 cycles Cycle number depends on the amount of template cdna and abundance of the target. * For Eppendorf Mastercyler ep realplex models 2, 2S, 4, and 4S: for the Silver Thermoblock, adjust the ramp rate to 26%; for the Aluminum Thermoblock, adjust the ramp rate to 35%. If using a Roche LightCycler 480, adjust the ramp rate to 1.6 C/s. Due to software requirements, the fluorescence detection step must be at least 30 s with the ABI PRISM 7000 or 34 s with the Applied Biosystems 7300 and If using the Roche LightCycler 480, use 45 cycles. 8. Place the plate/rotor-disc in the real-time cycler and start the cycling program. 9. Once the real-time PCR run has been completed, continue to Protocol: Data analysis using the C T method of relative quantification. 30 miscript Plant qpcr System Handbook 06/2014

31 Protocol: Data analysis using the C T method of relative quantification This protocol describes the steps for analysis of data from miscript mirna PCR Arrays. The first steps should be performed by the user. The later steps are performed by the GeneGlobe Data Analysis Center. The tools automatically perform quantification using the C T method of relative quantification and interpretation of the control wells. Results are presented in a tabular format, a scatter plot, a three-dimensional profile, and a volcano plot (when triplicates or greater are included). Important point before starting Text marked with a denotes instructions for 96-well and 384-well plates (formats A, C, D, E, F, and G); text marked with a denotes instructions for 100-well Rotor-Discs (format R). Procedure Steps performed by the user 1. Define the baseline. The baseline is the noise level in early cycles, where there is no detectable increase in fluorescence due to PCR products. Use the Linear View of the amplification plot to determine the earliest visible amplification. Set the baseline from cycle 2 to 2 cycles before the earliest visible amplification. Do not use greater than cycle 15. The number of cycles used to calculate the baseline can be changed and should be reduced if high template amounts are used. For more information regarding real-time PCR data output, refer to Appendix B, page 42. For the Rotor-Gene Q, we recommend using the Dynamic Tube setting along with the Slope Correct and/or Ignore First settings. For more information, refer to the Rotor-Gene Q User Manual. Note: Ensure that baseline settings are the same across all PCR runs associated with the same experiment to allow comparison of results. For this reason, we do not recommend using automatic data analysis settings. 2. Define the threshold. The threshold should be set using a logarithmic amplification plot so that the log-linear range of the curve can be easily identified. Using the Log View of the amplification plot, place the threshold above the background signal but within the lower half of the logmiscript Plant qpcr System Handbook 06/

32 linear range of the amplification plot. The threshold should never be set in the plateau phase. The absolute position of the threshold is less critical than its consistent position across PCR runs. Various PCR instruments (such as Applied Biosystems models 7500 and ViiA 7, and Agilent models Mx3005P and Mx3000P) may require adjustment of the default Manual C T threshold value of 0.2 to a lower value in order to analyze the data properly. Use a value of 0.02 as a starting point. For the Rotor-Gene Q, we recommend a C T threshold value of approximately 0.02 in order to analyze the data properly. Note: Ensure that threshold settings are the same across all PCR runs in the same analysis to allow comparison of results. 3. Export C T values according to the manual supplied with the real-time PCR instrument. 4. Access the GeneGlobe Data Analysis Center. Steps performed by data analysis software 5. All C T values reported as greater than 35 or as N/A (not detected) are changed to 35, or as greater than 33 or as N/A (not detected) are changed to 33. Therefore, any C T value equal to 35 or 33 is considered a negative call and is representative of less than 1 copy. 6. C T values of the positive PCR control wells (PPC) are examined. If the RNA sample is of high quality, the cycling program has been correctly run, and the thresholds have been correctly defined, the value of C T PPC should be 19±2 or 15±2 across all arrays or samples. 7. C T values of the miscript Plant Ligation Controls (miplc) and miscript Plant Reverse-Transcription Controls (miprc) are examined using the values for the positive PCR control (PPC) by calculating: ΔC T = AVG C T miplc AVG C T PPC ΔC T = AVG C T miprc AVG C T PPC If this value is less than 7, for miplc-ppc, then there is no apparent inhibition of the combined ligation/reverse-transcription reaction reaction. If this value is less than 7, for miprc-ppc, then there is no apparent inhibition of the reverse-transcription reaction. If this value is greater than 7 for either calculation, there is evidence of impurities that may have inhibited the reactions. See the Troubleshooting Guide, page miscript Plant qpcr System Handbook 06/2014

33 Note: The miplc and miprc calculations are specific for 96-well and 384- well (4 x 96, 4 sample) miscript mirna PCR Arrays. For 384-well (HC, 1 sample) and 4 x 96 miscript mirna PCR Arrays, as the cdna is divided among significantly more wells, a correction factor is introduced into the calculation according to the number of plates/rotor-discs that are being used. This corrects for the dilution of the miplc and miprc controls. The calculation is performed as shown in Table 11. Table 11. ΔCT (miplc or miprc-ppc) calculation for 384-well (HC, 1 sample) and 4 x 96 miscript mirna PCR Arrays Number of plates/ Rotor-Discs 384- well 96- well Rotor-Disc 100 Correction factor ΔC T (mirtc-ppc) calculation miplc or miprc (AVG C T 1.1) (AVG PPC C T ) miplc or miprc (AVG C T 2.1) (AVG PPC C T ) 8. ΔC T value for each mature mirna profiled in the plate is calculated using the formula ΔC T = C T mirna C T Normalizer. Note: Choose an appropriate normalizer for your sample. snorna/snrna control for normalization. Make sure that the selected controls are not influenced by the experimental conditions. Options for normalization include selecting up to six snorna/snrna controls, choosing an invariant mirna, or calculating a plate mean of expressed mirnas. 9. C T for each mirna across 2 miscript mirna PCR Arrays or 2 samples is calculated using the formula: C T = C T (sample 2) C T (sample 1) where sample 1 is the control sample and sample 2 is the experimental sample. 10. Fold-change for each gene from sample 1 to sample 2 is calculated as 2 (- CT). Optional: If the fold-change is greater than 1, the result may be reported as miscript Plant qpcr System Handbook 06/

34 a fold increase. For example, a mirna with a fold-change of 10 can be reported as a 10-fold increase. If the fold-change is less than 1, the negative inverse of the result may be reported as a fold decrease. For example, a mirna with a fold-change of 0.1 can be reported as 10 or a 10-fold decrease. 11. Fold-changes are presented by the data analysis tool in a variety of formats, including a tabular format, a scatter plot, a three-dimensional profile, and a volcano plot (when replicates are included). 34 miscript Plant qpcr System Handbook 06/2014

35 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise. For more information, see also the Frequently Asked Questions page at our Technical Support Center: The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information or protocols in this handbook or sample and assay technologies (for contact information, see back cover or visit Comments and suggestions Evidence of ligation/reverse-transcription (value of AVG C T miplc AVG C T PPC > 7) or poor reverse-transcription efficiency (value of AVG C T miprc AVG C T PPC > 7) a) Poor-quality RNA Check the A 260 :A 280 and A 260 :A 230 ratios of the RNA samples. Be sure to perform the dilutions for spectrophotometry in RNase-free 10 mm Tris Cl, ph 7.5. If necessary, repurify RNA with a spincolumn based clean up method, such as the mirneasy Mini Kit (cat. no ). b) Calculation did not include correction factor If using 384-well (HC, 1 sample) or 4 x 96 miscript mirna PCR Arrays, be sure to include the correction factors detailed in Table 10, page 30. Evidence of poor PCR amplification efficiency (AVG CT PPC varies by more than 2 across arrays and/or is greater than 21 for 96-well and 384-well plates or 17 for 100-well Rotor-Discs) a) Variation in instrument sensitivity b) HotStarTaq DNA Polymerase not activated with a hot start Different instruments have different levels of sensitivity. If an average C T PPC value of 19±2 for 96-well and 384-well plates or 15±2 for 100-well Rotor-Discs is difficult to obtain for the instrument used, the observed average C T PPC value should be acceptable as long as it does not vary by more than 2 cycles between arrays being compared. Be sure that the initial heat activation step at 95 C took place for 15 minutes, and that all other cycle parameters were performed according to the protocol. miscript Plant qpcr System Handbook 06/

36 c) Poor-quality RNA that may contain PCR inhibitors Comments and suggestions Check the A 260 :A 280 and A 260 :A 230 ratios of the RNA samples. Be sure to perform the dilutions for spectrophotometry in RNase-free 10 mm Tris Cl, ph 7.5. If necessary, repurify RNA with a spincolumn based clean up method, such as the mirneasy Mini Kit (cat. no ). No product, or product detected late in real-time PCR (indicative of problems occurring during ligation or reverse-transcription) a) Poor quality or incorrect amount of template RNA Check the concentration, integrity, and purity of the template RNA before starting the protocol. Mix well after thawing the template RNA. Even small amounts of RNases can affect synthesis of cdna and sensitivity in RT-PCR, particularly with small amounts of RNA. See Table 2, page 18 for recommended RNA amounts. b) RNA denatured Denaturation of the template RNA is not necessary. If denaturation was performed, the integrity of the RNA may be affected. c) Pipetting error or missing reagent when setting up ligation or reverse-transcription reaction d) Incorrect setup of ligation reaction e) 2x miscript Ligation Activator was not added to the ligation reaction f) Incubation temperature too high Check the pipets used for experimental setup. Mix all reagents well after thawing and repeat the reverse-transcription reaction. Be sure to set up the reaction at room temperature and thoroughly pipet up and down (at least 12 times) to mix. Be sure to add exactly 10 µl of 2x miscript Ligation Activator to each ligation reaction. Ligation should be carried out at 16ºC. Higher temperatures will reduce the activity of miscript Plant RNA Ligase. 36 miscript Plant qpcr System Handbook 06/2014

37 g) Incorrect setup of reverse-transcription reaction h) Too much ligation product transferred into reverse-transcription reaction i) Incubation temperature too high i) Incorrect dilution of synthesized cdna Comments and suggestions Be sure to set up the reaction on ice. Be sure to transfer exactly 4 µl ligation product into the reverse-transcription reaction. Reverse-transcription should be carried out at 37ºC. Higher temperatures will reduce the activity of miscript Plant Reverse Transcriptase. After cdna synthesis, cdna must be minimally diluted 11-fold. From there, maximally 1 µl of the diluted cdna can be used per qpcr reaction. See Table 5, page 21 for recommended cdna dilutions. No product, or product detected late in real-time PCR, or only primer-dimers detected (indicative of problems occurring during real-time PCR) a) PCR annealing time too short Use the annealing time specified in the protocol. b) PCR extension time too short c) Pipetting error or missing reagent when setting up PCR d) HotStarTaq DNA Polymerase not activated with a hot start Use the extension time specified in the protocol. Check the concentrations and storage conditions of reagents, including primers and cdna. Ensure that the cycling program includes the hot start activation step for HotStarTaq DNA polymerase; for details, check the protocol. e) No detection activated Check that fluorescence detection was activated in the cycling program. f) Wrong detection step Ensure that fluorescence detection takes place during the extension step of the PCR cycling program. miscript Plant qpcr System Handbook 06/

38 g) Wrong dye layer/filter chosen h) Insufficient starting template Comments and suggestions Ensure that the appropriate layer/filter is activated. Increase the amount of template cdna. No linearity in ratio of C T value/crossing point to log of the template amount a) Template amount too high Do not exceed maximum recommended amounts of template cdna. For details, see the protocol. b) Template amount too low Increase amount of template RNA. Varying fluorescence intensity a) Real-time cycler contaminated Decontaminate the real-time cycler according to the supplier s instructions. b) Real-time cycler no longer calibrated Recalibrate the real-time cycler according to the supplier s instructions. 38 miscript Plant qpcr System Handbook 06/2014

39 References QIAGEN maintains a large, up-to-date online database of scientific publications utilizing QIAGEN products. Comprehensive search options allow you to find the articles you need, either by a simple keyword search or by specifying the application, research area, title, etc. For a complete list of references, visit the QIAGEN Reference Database online at or contact QIAGEN Technical Services or your local distributor. Appendix A: Isolation of total RNA, including small RNAs, from plant tissues A generic protocol for RNA extraction from plants is difficult to suggest, as plants differ widely in their physical makeup and can contain large amounts of problematic material which can interfere with downstream analysis. The protocol below should be sufficient for simple green leaves, soft roots, and other plant parts. We recommend a literature search for publications detailing more specialized isolation procedures for your specific sample type, or call QIAGEN technical support for assistance. Equipment and reagents to be supplied by user mirneasy Mini Kit (cat. no ) TissueLyser II (cat. no ) TissueLyser Adapter Set 2 x 24 (cat. no ) Stainless Steel Beads, 5 mm (cat. no ) MaXtract High Density Tubes, 2 ml (cat. no ) Chloroform (without added isoamyl alcohol) Ethanol (70% and %); do not use denatured alcohol, which contains other substances such as methanol and methylethylketone Sterile, RNase-free pipet tips 1.5 ml or 2 ml microcentrifuge tubes Microcentrifuge(s) (with rotor for 2 ml tubes) for centrifugation at 4 C and at room temperature (15 25 C) Optional: RNase-Free DNase Set (cat. no ) miscript Plant qpcr System Handbook 06/

40 Important points before starting If using the mirneasy Mini Kit for the first time, read Important Notes in the mirneasy Mini Handbook. The TissueLyser II is recommended for optimal sample disruption and homogenization. As an alternative to the TissueLyser II, the TissueLyser LT or TissueRuptor can be used. For more information, visit This protocol is for isolation of total RNA from fresh tissues. Buffer RWT may form a precipitate upon storage. If necessary, redissolve by warming and then place at room temperature (15 25ºC). QIAzol Lysis Reagent and Buffer RWT contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach. See the Safety Information section in the mirneasy Mini Handbook. Except for phase separation (step A11), all protocol and centrifugation steps should be performed at room temperature. Things to do before starting Buffers RWT and RPE are supplied as concentrates. Before using for the first time, add the required volumes of ethanol (96 100%), as indicated on the bottle, to obtain a working solution. Procedure A1. Transfer a single TissueLyser stainless steel bead to a 2 ml centrifuge tube. A2. Place 25 mg fresh plant tissue in the 2 ml centrifuge tube containing the stainless steel bead. Note: 50 mg plant tissue can maximally be added. If there is a need to do 100 mg, do separate 50 mg isolations and combine at the aqueous phase. A3. Add 1 ml QIAzol Lysis Reagent and securely cap the tube(s). A4. Place the tube(s) in the TissueLyser Adapter Set 2 x 24. A5. Operate the TissueLyser for 1 min at Hz. Disassemble the adapter set, rotate the rack of tubes so that the tubes nearest to the TissueLyser are now outermost, and reassemble the adapter set. Operate the TissueLyser for another 1 min at Hz. The duration of disruption and homogenization depends on the tissue being processed and can be extended until no tissue debris is visible. A6. Place the tube on the benchtop at room temperature (15 25ºC) for 5 min. This step promotes dissociation of nucleoprotein complexes. 40 miscript Plant qpcr System Handbook 06/2014

41 A7. Immediately before use, pellet MaXtract High Density by centrifugation for s at 12,000 16,000 x g in a microcentrifuge. A8. Transfer homogenized lysate (from step A6) to the MaXtract High Density tube. A9. To the MaXtract High Density Tube, add 200 µl chloroform. Securely cap the tube and shake the tube vigorously for 15 s. Thorough mixing is important for subsequent phase separation. IMPORTANT: Do not vortex. A10. Place the MaXtract High Density Tube on the benchtop at room temperature for 2 3 min. A11. Centrifuge for 15 min at 12,000 x g at 4ºC. After centrifugation, heat the centrifuge up to room temperature (15 25ºC) if the same centrifuge will be used for the next centrifugation steps. After centrifugation, the sample separates into 3 phases: an upper, colorless, aqueous phase containing RNA; a white interphase (MaXtract High Density gel); and a lower, red, organic phase. A12. Transfer upper aqueous phase to a new tube. Add 1.5 volumes of 100% ethanol and mix thoroughly by pipetting up and down several times. Do not centrifuge. Continue without delay with Step A13. A13. Pipet up to 700 µl of the sample into an RNeasy Mini spin column in a 2 ml collection tube. Close the lid gently and centrifuge for 15 s at 8000 x g ( 10,000 rpm) at room temperature (15 20 ºC). Discard the flowthrough.* Reuse the collection tube in step A14. A14. Repeat step A13 using the remainder of the sample. Discard the flowthrough.* Reuse the collection tube in step A15. Optional: If performing optional on-column DNase digestion, follow steps B1 B4 (mirneasy Mini Kit Handbook, page 36) after performing this step. A15. Add 700 µl Buffer RWT into RNeasy Mini Spin column and centrifuge for 15 s at 8000 x g ( 10,000 rpm) to wash. Discard the flow-through.* Reuse the collection tube in step A16. A16. Pipet 500 µl Buffer RPE to column. Close lid and centrifuge for 15 s at 8000 x g ( 10,000 rpm) to wash the column. Discard flow-through. Reuse the collection tube in step A17. A17. Add another 500 µl Buffer RPE to column. Close lid gently and centrifuge for 2 min at 8000 x g ( 10,000 rpm) to dry the RNeasy Mini spin column membrane. The long centrifugation dries the spin column * Flow-through contains QIAzol Lysis Reagent and is therefore not compatible with bleach. See page 6 in the mirneasy Mini Kit Handbook for safety information. miscript Plant qpcr System Handbook 06/

42 membrane, ensuring that no ethanol is carried over during RNA elution. Residual ethanol may interfere with downstream reactions. Note: Following centrifugation, remove the RNeasy Mini spin column from the collection tube carefully so the column does not contact the flowthrough. Otherwise, carryover of ethanol will occur. A18. Place the RNeasy Mini spin column into a new 2 ml collection tube (not supplied), and discard the old collection tube with the flow-through. Centrifuge 1 min at full speed. Perform this step to eliminate any possible carryover of Buffer RPE or if residual flow-through remains on the outside of the RNeasy Mini spin column after step A17. A19. Transfer the RNeasy Mini spin column to a new 1.5 ml collection tube. Add 30 µl RNase-free water directly onto the RNeasy Mini spin column membrane. Close lid gently and centrifuge for 1 min at 8000 x g ( 10,000 rpm) to elute RNA. A20. Repeat Step A19 with a second volume of 20 µl RNase-free water. Elute into the same collection tube. Appendix B: Real-Time PCR data output and dissociation curve analysis In a typical amplification plot resulting from a real-time PCR reaction, fluorescence is plotted against the cycle count, producing sigmoidal-shaped plots (when using a linear scale). The threshold cycle (C T ) serves as a tool for calculation of the starting template amount in each sample. This is the cycle in which there is the first detectable increase in fluorescence. There may be variation in how determination of C T values is carried out depending on the realtime PCR cycler that is used. 42 miscript Plant qpcr System Handbook 06/2014

43 Figure 7. Amplification plot. Amplification plots showing increases in fluorescence from 2 samples (A and B). Sample A contains a greater amount of starting template than sample B. Figure 8. Typical amplification plot. Amplification plot after quantification of a range of amounts of a mature mirna. Real-time PCR was performed using the Rotor-Gene Q. Dissociation curve analysis A dissociation curve analysis of PCR product(s) may be optionally performed to aid in verifying their specificity and identity. Dissociation curve analysis is an analysis step built into the software of real-time cyclers. Follow instructions provided by the supplier. To carry out dissociation curve analysis, the temperature is increased very slowly from a low temperature (e.g., 65ºC) to a high temperature (e.g., 95ºC). miscript Plant qpcr System Handbook 06/

44 At low temperatures, PCR products are double-stranded, so SYBR Green I dye binds to them and fluorescence is high. However at high temperatures, PCR products are denatured, resulting in rapid decreases in fluorescence. The fluorescence is measured continuously as the temperature is increased and the fluorescence values are plotted against temperature. A curve is produced, because fluorescence decreases slightly through the lower end of the temperature range, but decreases much more rapidly at higher temperatures as the dissociation temperatures of nonspecific and specific PCR products are reached. The detection systems calculate the first derivatives of the curves, resulting in curves with peaks at the respective Tms (Figures 9 and 10). Curves with peaks at a T m lower than that of the specific PCR product indicate the formation of primer-dimers, while diverse peaks with different T m s or plateaus indicate production of nonspecific products or a smear. Figure 8. Dissociation curve analysis. Dissociation curve analysis of 2 samples (A and B). Sample A yields only 1 peak, resulting from the specific amplification product (primer-dimers not coamplified). Sample B shows a peak from the specific product and a peak at a lower temperature from amplification of primer-dimers. 44 miscript Plant qpcr System Handbook 06/2014

45 Figure 9. mirna dissociation curve. Dissociation curve analysis of a mature mirna PCR product showing a single peak from the specific amplification product. Dissociation curve analysis was performed using the Rotor-Gene Q. Appendix C: General remarks on handling RNA Handling RNA Ribonucleases (RNases) are very stable and active enzymes that generally do not require cofactors to function. Since RNases are difficult to inactivate and even minute amounts are sufficient to degrade RNA, do not use any plasticware or glassware without first eliminating possible RNase contamination. Care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after the purification procedure. In order to create and maintain an RNase-free environment, the following precautions must be taken during pretreatment and use of disposable and nondisposable vessels and solutions while working with RNA. General handling Proper microbiological, aseptic technique should always be used when working with RNA. Hands and dust particles may carry bacteria and molds and are the most common sources of RNase contamination. Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase miscript Plant qpcr System Handbook 06/

46 contamination from the surface of the skin or from dusty laboratory equipment. Change gloves frequently and keep tubes closed whenever possible. Keep purified RNA on ice. To remove RNase contamination from bench surfaces, nondisposable plasticware, and laboratory equipment (e.g., pipets and electrophoresis tanks), use of RNaseKiller (cat. no ) from 5 PRIME ( is recommended. RNase contamination can alternatively be removed using general laboratory reagents. To decontaminate plasticware, rinse with 0.1 M NaOH, 1 mm EDTA * followed by RNase-free water (see Solutions, page 46), or rinse with chloroform* if the plasticware is chloroform-resistant. To decontaminate electrophoresis tanks, clean with detergent (e.g., 0.5% SDS),* rinse with RNase-free water, rinse with ethanol (if the tanks are ethanolresistant), and allow to dry. Disposable plasticware The use of sterile, disposable polypropylene tubes is recommended throughout the procedure. These tubes are generally RNase-free and do not require pretreatment to inactivate RNases. Glassware Glassware should be treated before use to ensure that it is RNase-free. Glassware used for RNA work should be cleaned with a detergent, thoroughly rinsed, and oven baked at 240ºC for 4 hours or more (overnight, if more convenient) before use. Autoclaving alone will not fully inactivate many RNases. Alternatively, glassware can be treated with DEPC* (diethyl pyrocarbonate), as described in Solutions below. Solutions Note: QIAGEN solutions, such as 10x miscript Ligation Buffer, 10x miscript Plant Adaptor, 2x miscript Ligation Activator, miscript Plant RNA Ligase, 5x miscript Plant RT Buffer, 10x miscript Plant RT Nucleics, miscript Plant Reverse Transcriptase, and RNase-Free Water are guaranteed RNase-free without using DEPC treatment and are therefore free of any DEPC contamination. Solutions (water and other solutions) should be treated with 0.1% DEPC. DEPC is a strong, but not absolute, inhibitor of RNases. DEPC is commonly used at a concentration of 0.1% to inactivate RNases on glass or plasticware or to create * When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate material data sheets (MSDSs), available from the product supplier. 46 miscript Plant qpcr System Handbook 06/2014

47 RNase-free solutions and water. DEPC inactivates RNases by covalent modification. Add 0.1 ml DEPC to 100 ml of the solution to be treated and shake vigorously to bring the DEPC into solution. Let the solution incubate for 12 hours at 37ºC. Autoclave for 15 minutes to remove any trace of DEPC. DEPC will react with primary amines and cannot be used directly to treat Tris* buffers. DEPC is highly unstable in the presence of Tris buffers and decomposes rapidly into ethanol and CO 2. When preparing Tris buffers, treat water with DEPC first, and then dissolve Tris to make the appropriate buffer. Trace amounts of DEPC will modify purine residues in RNA by carbethoxylation. Carbethoxylated RNA is translated with very low efficiency in cell-free systems. However, its ability to form DNA:RNA or RNA:RNA hybrids is not seriously affected unless a large fraction of the purine residues have been modified. Residual DEPC must always be eliminated from solutions or vessels by autoclaving or heating to 100ºC for 15 minutes. Appendix D: Preparation, quantification, and storage of RNA Storage of RNA Purified RNA may be stored at 15 C to 30 C or 65 C to 90 C in RNasefree water. Under these conditions, no degradation of RNA is detectable after 1 year. Quantification of RNA The concentration of RNA should be determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer, e.g. the QIAxpert (see Spectrophotometric quantification of RNA below). For small amounts of RNA, however, it may be difficult to determine amounts photometrically. Small amounts of RNA can be quantified using an Agilent 2100 Bioanalyzer, quantitative RT-PCR, or fluorometric quantification. When purifying RNA from particularly small samples (e.g., laser-microdissected samples, or from plasma or serum), quantitative, real-time RT-PCR should be used for quantification. Spectrophotometric quantification of RNA To ensure significance, A260 readings should be greater than An absorbance of 1 unit at 260 nm corresponds to 44 μg of RNA per ml (A260=1 44 μg/ml). This relation is valid only for measurements at a neutral ph. Therefore, if it is necessary to dilute the RNA sample, this should be done in miscript Plant qpcr System Handbook 06/

48 a buffer with neutral ph), the ratio between the absorbance values at 260 and 280 nm gives an estimate of RNA purity. As discussed below (see Purity of RNA, page 48), the ratio between the absorbance values at 260 and 280 nm gives an estimate of RNA purity. When measuring RNA samples, be certain that cuvettes are RNase-free, especially if the RNA is to be recovered after spectrophotometry. This can be accomplished by washing cuvettes with 0.1 M NaOH, 1 mm EDTA, followed by washing with RNase-free water (see Solutions, page 46). Use the buffer in which the RNA is diluted to zero the spectrophotometer. An example of the calculation involved in RNA quantification is shown below: Volume of RNA sample = 100 μl Dilution = 10 μl of RNA sample μl of 10 mm Tris Cl,* ph 7.0 (1/50 dilution) Measure absorbance of diluted sample in a 1 ml cuvette (RNase-free) A260 = 0.2 Concentration of RNA sample = 44 μg/ml x A260 x dilution factor Total amount Purity of RNA = 44 μg/ml x 0.2 x 50 = 440 μg/ml = concentration x volume in milliliters = 440 μg/ml x 0.1 ml = 44 μg of RNA The ratio of the readings at 260 nm and 280 nm (A260/A280) provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV spectrum, such as protein. However, the A260/A280 ratio is influenced considerably by ph. Since water is not buffered, the ph and the resulting A260/A280 ratio can vary greatly. Lower ph results in a lower A260/A280 When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate material data sheets (MSDSs), available from the product supplier. 48 miscript Plant qpcr System Handbook 06/2014

49 ratio and reduced sensitivity to protein contamination. For accurate values, we recommend measuring absorbance in 10 mm Tris Cl, ph 7.5. Pure RNA has an A260/A280 ratio of in 10 mm Tris Cl, ph 7.5. Always be sure to calibrate the spectrophotometer with the same solution used for dilution. For determination of RNA concentration, however, we recommend dilution of the sample in a buffer with neutral ph since the relationship between absorbance and concentration (A260 reading of 1 = 44 μg/ml RNA) is based on an extinction coefficient calculated for RNA at neutral ph (see Quantification of RNA, page 47). DNA contamination No currently available purification method can guarantee that RNA is completely free of DNA, even when it is not visible on an agarose gel. While mirneasy Kits will remove the vast majority of cellular DNA, trace amounts may still remain, depending on the amount and nature of the sample. However, serum, plasma, and other cell-free body fluids contain very little DNA. For analysis of very low-abundance targets, any interference by residual DNA contamination can be detected by performing real-time RT-PCR control experiments in which no reverse transcriptase is added prior to the PCR step. Integrity of RNA The integrity and size distribution of total RNA purified with mirneasy Kits can be checked by denaturing agarose gel electrophoresis and ethidium bromide staining or by using the QIAxcel system or Agilent 2100 Bioanalyzer. The respective ribosomal RNAs should appear as sharp bands or peaks. The apparent ratio of 28S rrna to 18S rrna should be approximately 2:1. If the ribosomal bands or peaks of a specific sample are not sharp, but appear as a smear towards smaller sized RNAs, it is likely that the sample suffered major degradation either before or during RNA purification. The QIAxcel and Agilent 2100 Bioanalyzer also provide an RNA Integrity Score (RIS) or RNA Integrity Number (RIN) as useful measures of RNA integrity. Ideally, the RIS/RIN should be close to 10, but in many cases (particularly with tissue samples), RNA quality is greatly influenced by how well the original sample was preserved. Cell-free RNA from serum or plasma contains high proportions of small RNAs of less than 100 nucleotides. Even though full-length mrna and intact ribosomal Wilfinger, W.W., Mackey, M., and Chomczynski, P. (1997) Effect of ph and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques 22, 474. Values up to 2.3 are routinely obtained for pure RNA (in 10 mm Tris Cl, ph 7.5) with some spectrophotometers. miscript Plant qpcr System Handbook 06/

50 RNA can be isolated from EVs, the rrna peaks are not always visible on the Bioanalyzer. RIS or RIN are therefore not useful as indicators of RNA integrity for cell-free RNA from EVs (or from total plasma or serum). 50 miscript Plant qpcr System Handbook 06/2014

51 Ordering Information Product Contents Cat. no. miscript Plant RT Kit (12) For 12 cdna synthesis reactions: 10x miscript Ligation Buffer, 10x miscript Plant Adaptor, 2x miscript Ligation Activator, miscript Plant RNA Ligase, 5x miscript Plant RT Buffer, 10x miscript Plant RT Nucleics, miscript Plant Reverse Transcriptase, RNase-Free Water miscript Plant RT Kit (50) miscript Plant RT Kit (96) miscript SYBR Green PCR Kit (200) miscript SYBR Green PCR Kit (1000) miscript SYBR Green PCR Kit (2000) miscript Primer Assay (100) For 50 cdna synthesis reactions: 10x miscript Ligation Buffer, 10x miscript Plant Adaptor, 2x miscript Ligation Activator, miscript Plant RNA Ligase, 5x miscript Plant RT Buffer, 10x miscript Plant RT Nucleics, miscript Plant Reverse Transcriptase, RNase-Free Water For 96 cdna synthesis reactions: 10x miscript Ligation Buffer, 10x miscript Plant Adaptor, 2x miscript Ligation Activator, miscript Plant RNA Ligase, 5x miscript Plant RT Buffer, 10x miscript Plant RT Nucleics, miscript Plant Reverse Transcriptase, RNase-Free Water For 200 reactions: QuantiTect SYBR Green PCR Master Mix, miscript Universal Primer For 1000 reactions: QuantiTect SYBR Green PCR Master Mix, miscript Universal Primer For 2000 reactions: QuantiTect SYBR Green PCR Master Mix, miscript Universal Primer 10x miscript Primer Assay (contains one mirna-specific primer) Varies miscript Plant qpcr System Handbook 06/

52 Product Contents Cat. no. miscript mirna PCR Array Array of assays for a pathway, disease, or gene family; available in 96-well, 384- well, or Rotor-Disc 100 format Varies RT 2 PCR Array Loading Reservoir 384EZLoad Covers Related Products mirneasy Mini Kit (50) 12 x 5 ml capacity, irradiation-sterilized loading reservoirs Pack of 4 color-coded covers for loading 384-well plates For 50 preps: 50 RNeasy Mini Spin Columns, Collection Tubes (1.5 ml and 2 ml), QIAzol Lysis Reagent, RNase-Free Reagents and Buffers MaXtract High Density Tubes, 2 ml 200 x 2ml MaXtract High Density Tubes RNase-Free DNase Set (50) 1500 units RNase-free DNase I, RNase- Free Buffer RDD, and RNase-Free Water for 50 RNA minipreps TissueLyser II Universal laboratory mixer mill disruptor TissueLyser Adapter Set 2 x 24 TissueLyser Adapter Set 2 x 96 Stainless Steel Beads, 5 mm (200) 2 sets of Adapter Plates and 2 racks for use with 2 ml microcentrifuge tubes on the TissueLyser II 2 sets of Adapter Plates for use with Collection Microtubes (racked) on the TissueLyser II Stainless Steel Beads, suitable for use with TissueLyser systems For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at or can be requested from QIAGEN Technical Services or your local distributor. 52 miscript Plant qpcr System Handbook 06/2014

53 Notes miscript Plant qpcr System Handbook 06/

54 Notes 54 miscript Plant qpcr System Handbook 06/2014

55 Trademarks: Trademarks: QIAGEN, QIAzol, QIAgility, HotStarTaq, QuantiTect, RNeasy, Rotor-Gene, Rotor-Disc, MinElute (QIAGEN Group); PAXgene (PreAnalytiX GmbH); Roche, LightCycler, TaqMan (Roche Group); ABI PRISM, Applied Biosystems, StepOnePlus, ViiA (Applera Corporation or its subsidiaries); Eppendorf, Mastercycler (Eppendorf AG); Stratagene, Mx3005P, Mx3000P, Mx4000 (Stratagene); Bio-Rad, icycler, Chromo4, CFX96, DNA Engine Opticon, CFX384, iq, MyiQ (Bio-Rad Laboratories, Inc.); GeneAmp, ROX, SYBR (Life Technologies Corporation); SmartCycler (Cepheid); Excel (Microsoft, Inc.). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law. Limited License Agreement for miscript Plant qpcr System Use of this product signifies the agreement of any purchaser or user of the product to the following terms: 1. The product may be used solely in accordance with the protocols provided with the product and this handbook and for use with components contained in the kit only. QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included within this kit except as described in the protocols provided with the product, this handbook, and additional protocols available at Some of these additional protocols have been provided by QIAGEN users for QIAGEN users. These protocols have not been thoroughly tested or optimized by QIAGEN. QIAGEN neither guarantees them nor warrants that they do not infringe the rights of third-parties. 2. Other than expressly stated licenses, QIAGEN makes no warranty that this kit and/or its use(s) do not infringe the rights of third-parties. 3. This kit and its components are licensed for one-time use and may not be reused, refurbished, or resold. 4. QIAGEN specifically disclaims any other licenses, expressed or implied other than those expressly stated. 5. The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and/or its components. For updated license terms, see QIAGEN, all rights reserved.

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