USER GUIDE. Ovation PART NO One-Direct System

Transcription

1 USER GUIDE Ovation PART NO One-Direct System

2 Patents, Licensing and Trademarks NuGEN Technologies, Inc. All rights reserved. The Encore, Ovation and Applause families of products and methods of their use are covered by several issued U.S. and International patents and pending applications ( NuGEN, Ovation, SPIA, Ribo- SPIA, Applause, Encore, Prelude, Mondrian and Imagine More From Less are trademarks or registered trademarks of NuGEN Technologies, Inc. Other marks appearing in these materials are marks of their respective owners. The purchase of this product conveys to the buyer the limited, non-exclusive, non-transferable right (without the right to modify, reverse engineer, resell, repackage or further sublicense) under these patent applications and any patents issuing from these patent applications to use this product and methods, accompanying this user guide, for research and development purposes solely in accordance with the intended use described and the written instructions provided in this user guide. No license to make or sell products by use of this product is granted to the buyer whether expressly, by implication, by estoppels or otherwise. In particular, the purchase of this product does not include or carry any right or license to use, develop or otherwise exploit this product commercially and no rights are conveyed to the buyer to use the product or components of the product for purposes including commercial services or clinical diagnostics. For information on purchasing a license to the NuGEN patents for uses other than in conjunction with this product or to use this product for purposes other than research, please contact NuGEN Technologies, Inc., 201 Industrial Road, Suite 310, San Carlos, CA Phone or ; FAX or Warranty NuGEN warrants that this product meets the performance standards described in the Company s product and technical literature for a period of six months from the date of purchase, provided that the product is handled and stored according to published instructions, and that the product is not altered or misused. If the product fails to meet these performance standards, NuGEN will replace the product free of charge or issue a credit for the purchase price. NuGEN s liability under this warranty shall not exceed the purchase price of the product. NuGEN shall assume no liability for direct, indirect, consequential or incidental damages arising from the use, results of use or inability to use its products. NuGEN reserves the right to change, alter or modify any product to enhance its performance and design. NuGEN s products are developed, designed and sold FOR RESEARCH USE ONLY. This product is not to be used for diagnostic or therapeutic purposes, nor is it to be administered to humans or animals. Except as expressly set forth herein, no right to modify, reverse engineer, distribute, offer to sell or sell NuGEN s product is conveyed or implied by buyer s purchase of this NuGEN product. The buyer agrees to use NuGEN products accompanying the product insert in accordance with the intended use and the written instructions provided.

3 Table of Contents Contents I. Introduction... 1 A. Background... 1 B. How the Ovation One-Direct System Works... 1 C. Performance Specifications... 2 D. Quality Control... 2 E. Storage and Stability... 2 F. Material Safety Data Sheet (MSDS)... 3 II. Kit Components... 4 A. Reagents Provided... 4 B. Additional Equipment, Reagents and Labware... 5 III. Planning the Experiment... 7 A. Input RNA Requirements... 7 B. Using RNase-free Techniques... 9 C. RNA Storage... 9 D. Amplified cdna Storage... 9 IV. Protocol A. Overview B. Protocol Notes C. Agencourt RNAClean XP Purification Beads D. Programming the Thermal Cycler E. First Strand cdna Synthesis F. Second Strand cdna Synthesis G. Purification of cdna H. SPIA Amplification I. Post-SPIA Modification J. Purification of Amplified cdna Protocol K. Measuring cdna Product Yield and Purity V. Technical Support VI. Appendix A. Considerations for Microarray Analysis B. Performing Quantitative PCR on Amplified cdna C. Alternative Purification Protocols for Amplified cdna D. Quality Control of Amplified cdna Product E. DNase Treatment of RNA F. Preventing Non-specific Amplification G. Frequently Asked Questions (FAQs) H. Update History... 35

4 I. Introduction A. Background The Ovation One-Direct System provides a fast and simple method for preparing amplified cdna for global gene expression analysis directly from a cell lysate prepared from one or more cells, or purified total RNA samples as small as 10 pg. Amplification is initiated at the 3 end as well as randomly throughout the whole transcriptome in the sample. This feature, along with highly refined amplification chemistry, makes the Ovation One-Direct System ideal for amplification of the smallest biological samples down to the single-cell level. The amplified product of the Ovation One-Direct System is optimized for robust transcript detection using microarrays or real-time quantitative PCR (qpcr). The Ovation One-Direct System can be used with Affymetrix GeneChip arrays, Agilent Dual-Mode Gene Expression microarrays or Illumina Whole-Genome Expression BeadChips utilizing the appropriate NuGEN fragmentation and labeling modules and protocols. Reagents for fragmentation and labeling are not included in the Ovation One-Direct System and will need to be purchased separately. For details please visit the NuGEN website at The Ovation One-Direct System is powered by Ribo-SPIA technology, a rapid, simple and sensitive RNA amplification process developed by NuGEN. Using Ribo-SPIA technology and starting with a cell lysate from one or more cells, or 10 to 500 pg purified total RNA, microgram quantities of cdna can be prepared in approximately 8 hours. The Ovation One-Direct System (Part No. 3500) provides optimized reagent mixes and a protocol to process twelve RNA amplifications. Control RNA is not provided with the Ovation One-Direct System but we strongly recommend the use of a control RNA when using this product due to the inherent challenges in working with ultra-small biological samples. B. How the Ovation One-Direct System Works The Ovation One-Direct System utilizes Ribo-SPIA technology that produces amplified cdna from total RNA (see Figure 1). 1. Generation of First Strand cdna (1.5 hours) First strand cdna is prepared from a cell lysate or total RNA sample using a unique first strand DNA/RNA chimeric primer mix and reverse transcriptase (RT). The primers have a DNA portion that hybridizes either to the 5 portion of the poly (A) sequence or randomly across the transcript. RT extends the 3 DNA end of each primer generating first strand cdna. The resulting cdna/mrna hybrid molecule contains a unique RNA sequence at the 5 end of the cdna strand. 2. Generation of a DNA/RNA Heteroduplex Double Stranded cdna (2 hours) Fragmentation of the mrna within the cdna/mrna complex creates priming sites for DNA polymerase to synthesize a second strand, which includes DNA complementary to the 5 unique sequence from the first strand chimeric primers. The result is a double stranded cdna with a unique DNA/RNA heteroduplex at one end. 1 Ovation One-Direct System

5 I. Introduction 3. SPIA Amplification (2 hours) SPIA amplification is an isothermal DNA amplification process developed by NuGEN. The process uses a SPIA DNA/RNA chimeric primer, DNA polymerase and RNase H in a homogeneous isothermal assay that provides highly efficient amplification of DNA sequences. RNase H degrades RNA in the DNA/RNA heteroduplex at the 5 end of the first cdna strand. This exposes a DNA sequence that is available for binding the SPIA DNA/RNA chimeric primer. DNA polymerase initiates replication at the 3 end of the primer, displacing the existing forward strand. The RNA portion at the 5 end of the newly synthesized strand is again removed by RNase H, exposing the unique priming site for initiation of the next round of cdna synthesis. The process of SPIA DNA/RNA primer binding, DNA replication, strand displacement and RNA cleavage is repeated, resulting in rapid accumulation of cdna with sequence complementary to the original mrna. 4. Post-SPIA Modification (2 hours) The Post-SPIA Modification process completes the One-Direct amplification process. The first step allows the random primers to anneal to the single-stranded, anti-sense cdna target. The second step utilizes DNA polymerase to extend the annealed primers and generate sense-target cdna, producing targets appropriate for either sense or anti-sense array. C. Performance Specifications The Ovation One-Direct System synthesizes microgram quantities of amplified cdna starting with cell lysate or total cellular RNA input amounts of 10 pg to 500 pg. In approximately 7.5 hours, the system produces sufficient cdna for fragmentation and labeling, and subsequent hybridization to a microarray. Alternatively, the amplified product may be used for qpcr reactions. When used with intact input RNA the size of the majority of the cdna products produced by the amplification process is between 50 bases and 500 bases. D. Quality Control Each Ovation One-Direct System lot is tested to meet specifications of yield, qpcr and array performance. E. Storage and Stability Store First Strand and SPIA Primer at 80 C. Store the RNAClean XP Beads at 4 C The Ovation One-Direct System is shipped on dry ice and should be unpacked immediately upon receipt. Note: This product contains components with multiple storage temperatures. The vials labeled First Strand Primer Mix (blue: A1) and SPIA Primer Mix (red: C1) should be removed from the shipping carton upon delivery and stored separately at 80 C. The vial labeled Agencourt RNAClean XP Beads (clear cap) should be removed from the top of the shipping carton upon delivery and stored at 4 C. 2 Ovation One-Direct System

6 I. Introduction All remaining components should be stored at 20 C on the internal shelves of a freezer without a defrost cycle. After the first thaw, the Direct Lysis Buffer should be stored at 4 C. Do not refreeze the Lysis Buffer. The Ovation One-Direct System has been tested to perform to specifications for up to six freeze/thaw cycles. Kits handled and stored according to the above guidelines will perform to specifications for at least six months. We have not yet established long-term storage conditions for the Ovation One-Direct System. F. Material Safety Data Sheet (MSDS) An MSDS for this product is available on the NuGEN website at 3 Ovation One-Direct System

7 II. Kit Components A. Reagents Provided Table 1. First Strand cdna Reagents COMPONENT PART NUMBER VIAL CAP VIAL NUMBER First Strand Primer Mix S01250 Blue A1 ver 5 First Strand Buffer Mix S01248 Blue A2 ver 5 First Strand Enzyme Mix S01249 Blue A2 ver 3 Table 2. Second Strand cdna Reagents COMPONENT PART NUMBER VIAL CAP VIAL NUMBER Second Strand Buffer Mix S01176 Yellow B1 ver 3 Second Strand Enzyme Mix S01126 Yellow B2 ver 2 Table 3. SPIA Reagents COMPONENT PART NUMBER VIAL CAP VIAL NUMBER SPIA Primer Mix S01251 Red C1 ver 7 SPIA Buffer Mix S01164 Red C2 ver 5 SPIA Enzyme Mix S01165 Red C3 ver 5 4 Ovation One-Direct System

8 II. Kit Components Table 4. Post-SPIA Modification Reagents COMPONENT PART NUMBER VIAL CAP VIAL NUMBER Primer Mix S01252 Red E1 ver 1 Buffer Mix S01253 Red E2 ver 2 Enzyme Mix S01254 Red E3 ver 1 Table 5. Additional Reagents COMPONENT PART NUMBER VIAL CAP VIAL NUMBER Nuclease-free Water S01001 Green D1 Direct Lysis Buffer S01255 Clear Agencourt RNAClean XP Beads S01307 Clear Note: The reagents in the Ovation One-Direct System product are similar to reagents in NuGEN s other kits. However, unless the part numbers are identical, these reagents do not have exactly the same composition and therefore are not interchangeable. Do not exchange reagents between different kits as it will adversely affect performance. B. Additional Equipment, Reagents and Labware Required Materials Equipment -- Microcentrifuge for individual 1.5 ml and 0.5 ml tubes -- Microcentrifuge or centrifuge for individual 0.2 ml tubes, strip tubes and PCR plates µl pipette, 2 20 µl pipette, µl pipette, µl pipette -- Vortexer -- Thermal cycler with 0.2 ml tube heat block, heated lid, and 100 µl reaction capacity -- Appropriate spectrophotometer and cuvettes, or Nanodrop UV-Vis Spectrophotometer 5 Ovation One-Direct System

9 II. Kit Components Reagents -- Ethanol (Sigma-Aldrich, Cat. # E7023), for purification steps Supplies and Labware -- Nuclease-free pipette tips -- Low-retention microcentrifuge tubes (SafeSeal Low Binding 0.65 ml Microcentrifuge Tubes, Sorenson Biosciences, Inc., Cat. #11300) ml and 0.5 ml RNase-free microcentrifuge tubes ml individual thin-wall PCR tubes or 8 X 0.2 ml strip PCR tubes or 0.2 ml thin-wall PCR plate -- Magnetic separation device -- QIAGEN MinElute Spin Columns (Cat. #28204) -- Disposable gloves -- Kimwipes -- Ice bucket Optional materials -- Agilent 2100 bioanalyzer or materials and equipment for electrophoretic analysis of RNA -- Real-time PCR system -- Decontamination solutions such as RNaseZap (Ambion, Cat.# AM9780) and DNA-OFF (MP Biomedicals, Cat.# QD0500) To Order: Ambion Inc., Beckman Coulter, MP Biomedicals, New England BioLabs, QIAGEN Inc., Sigma-Aldrich, Inc., USB Corporation, Zymo Research, Sorenson Biosiences, 6 Ovation One-Direct System

10 III. Planning the Experiment A. Input RNA Requirements 1. RNA Quantity Ovation One-Direct is designed to use lysates from single cells, or cell pellets as input without further purification using the Direct Lysis Buffer included in the kit. Given that the amount of total RNA contained in a single cell varies widely based on cell type and other factors, some experimentation may be necessary to determine the optimum input cell numbers and working limits for a given sample type. Purified total RNA samples can also be used as input. We have successfully tested inputs in the range from 10 pg to 500 pg. 2. RNA Purity Purified total RNA samples must be free of contaminating proteins and other cellular material, organic solvents (including phenol and ethanol) and salts used in many RNA isolation methods. Use of a commercially available system for preparing small amounts of RNA that does not require organic solvents is recommended. If a method such as TRIzol is used, we recommend employing an additional column purification step after isolation in order to remove any residual organics. One measure of RNA purity is the ratio of absorbance readings at 260 and 280 nm. The A260:A280 ratio for RNA samples of acceptable purity should be in excess of 1.8. RNA samples with lower ratios may result in poor amplification. 3. RNA Integrity The Ovation One-Direct System is designed to use cell lysate samples prepared with Direct Lysis Buffer as input. Purified total RNA samples of high molecular weight with little or no evidence of degradation will also amplify very well with this product. RNA integrity can be determined using the Agilent 2100 Bioanalyzer, RNA 6000 Nano LabChip or RNA 6000 Pico LabChip. The RNA Integrity Number (RIN) calculation, available in the Bioanalyzer 2100 Expert Software, provides an index of RNA quality that can be helpful in triaging purified RNA samples of varying integrity prior to amplification. While it is impossible to guarantee satisfactory results with all degraded samples, the One-Direct System may work with many samples that are moderately or even severely degraded. 7 Ovation One-Direct System

11 III. Planning the Experiment Figure 1. This continuum of RNA quality shows Bioanalyzer traces of three different RNAs with varying levels of degradation. High-quality RNAs have been shown to amplify robustly with this kit. Use of lower quality mrnas may result in lower amplification yields. RNA Quality Continuum Poor Quality RIN=2.4 Moderate Quality RIN=6.7 Good Quality RIN= User Quality Control Guidelines for RNA samples The inclusion of positive control RNA samples is an essential tool in evaluating the success of an amplification experiment. Due to the limited sample quantities and the nature of the direct lysis process, it is impossible to determine the quantity and integrity of RNA. In the absence of successful positive control RNA amplification it will be difficult or impossible to troubleshoot amplification issues. 5. DNase Treatment DNase treatment is not necessary when using cell lysate samples prepared using the Direct Lysis Buffer. The use of DNase-treatment is highly recommended when using purified RNA samples. Contaminating genomic DNA will interfere with accurate quantitation of RNA samples, and may negatively impact detection sensitivity and data quality. See Appendix E for examples of DNase treatment protocols that have been used successfully with Ovation systems. 6. Carrier use for RNA isolation Carriers are not required when using cell lysate samples prepared with the Direct Lysis Buffer. If you are using purified total RNA samples, we strongly recommend against the use of yeast trna, because it has been shown to produce cdna product in first strand synthesis. We also advise against the use of glycogen in RNA isolation as it inhibits reverse transcription. For the latest information regarding other carriers, contact NuGEN s technical services team. 8 Ovation One-Direct System

12 III. Planning the Experiment B. Using RNase-free Techniques RNase contamination through reagents and work environment will lead to experimental failure. Follow these guidelines to minimize contamination: 1. Wear disposable gloves and change them frequently. 2. Avoid touching surfaces or materials that could introduce RNases. 3. Use reagents provided. Substitutions may introduce RNases. 4. Clean and decontaminate work areas and instruments, including pipettes, with commercially available decontamination reagents, such as RNaseZap and DNA-OFF. 5. Use only new RNase-free pipette tips and microcentrifuge tubes. 6. Use a work area specifically designated for RNA work and do not use other high copy number materials in the same area. C. RNA Storage RNA samples for use with the Ovation One-Direct System must be stored at 80 C. Avoid frequent freeze/thaw cycles of RNA as shearing may result. D. Amplified cdna Storage The amplified cdna produced by the Ovation One-Direct System may be stored at 20 C. 9 Ovation One-Direct System

13 IV. Protocol A. Overview The Ribo-SPIA amplification process used in the Ovation One-Direct System is performed in four stages: 1. First strand cdna synthesis 1.5 hours 2. Second strand cdna synthesis and purification 2 hours 3. SPIA isothermal linear amplification 2 hours 4. Post-SPIA modification and purification 2 hours Total time to prepare amplified library ~7.5 hours Ovation One-Direct System components are color coded, with each color linked to a specific stage of the process. Performing each stage requires making a master mix then adding it to the reaction, followed by incubation. Master mixes are prepared by mixing components provided for that stage. The Ovation One-Direct System may be used as a method of whole-transcriptome amplification prior to qpcr. Although for qpcr applications it is not absolutely necessary to purify the amplified cdna, we recommend proceeding with purification of cdna immediately after SPIA especially if you plan to mass normalize qpcr input. Spectrophotometric quantitation of unpurified amplification products will result in artificially high readings due to reaction components present in the sample. The cdna must be purified following amplification if you intend to use the cdna for fragmentation and labeling using a validated NuGEN product or protocol. B. Protocol Notes When working with very small, picogram amounts of RNA we strongly recommend the use of low retention tips and tubes for storage and diluting the samples, in order to reduce the loss of RNA samples due to adhesion to polypropylene surfaces. The first time you set up an amplification reaction, use a control RNA at an input level well above the minimum recommended amount. This allows you to establish a baseline of performance and provides the opportunity to become familiar with the bead purification step. This step is especially prone to handling variability in using the magnet plate so a practice run with the plate is also highly recommended. It is important to set up no fewer than 4 reactions at a time. This will ensure that you are not pipetting very small volumes (see the second strand synthesis section) below the effective range of air displacement pipetting technologies. For this reason, setting up fewer than 4 reactions can lead to poor performance. Thaw components used in each step and immediately place them on ice as indicated in the user guide instructions. It is best not to thaw reagents for all steps at once. The reagent color coding can be a guideline for appropriate reagent grouping. 10 Ovation One-Direct System

14 IV. Protocol Always keep thawed reagents and reaction tubes on ice unless otherwise instructed. After thawing and mixing buffer mixes, in rare instances a precipitate is observed. It is important that it be re-dissolved completely prior to use. You may gently warm the buffer mix for 2 minutes at room temperature followed by brief vortexing. Do not warm any enzyme or primer mixes. When placing small amounts of reagents into the reaction mix, pipet up and down several times to ensure complete transfer. When instructed to pipet mix, gently aspirate and dispense a volume that is at least half of the total volume of the reaction mix. Always allow the thermal cycler to reach the initial incubation temperature prior to placing the tubes or plates in the block. When preparing master mixes, use the minimal amount of extra material to ensure 12 reactions in the kit. Typically an overage factor of 10% is acceptable. For example, if making a master mix for 6 reactions use a factor of 6.6x when calculating the master mix volumes. Components and reagents from other Ovation System products should not be used with this product. Use only fresh ethanol stocks to make 70% ethanol used in the post-second strand bead purification (Section G), 80% ethanol for washes in the amplified cdna purification protocols (Section J, Appendix C) and ethanol washes for DNase Treatment and clean-up of RNA (Appendix E). Make the ethanol mixes fresh as well. Lower concentrations of ethanol in wash solutions will result in loss of yield as the higher aqueous content will dissolve the cdna and wash it off the beads or column. C. Agencourt RNAClean XP Purification Beads Tips and Notes There are significant modifications to the Agencourt RNAClean XP beads standard procedure; therefore, you must follow the procedures outlined in this user guide for the use of these beads with the Ovation One-Direct System. However, you may review the Agencourt user guide to become familiar with the manufacturer s recommendations, at the following website: RNACleanXPProtocol_001298v001.pdf The bead purification processes used in this kit consist of the following steps: 1. Binding of cdna to RNAClean XP beads 2. Magnetic separation of beads from supernatant 3. Ethanol wash of bound beads to remove contaminants 11 Ovation One-Direct System

15 IV. Protocol 4. Elution of bound cdna from beads Figure 2. Agencourt RNAClean XP Beads process overview. 1. Binding 2. Separation 3. Ethanol Wash 4. Elution Magnet Magnet Magnet Reproduced from original picture from Agencourt/Beckman Coulter Genomics Additional Tips and Notes Remove beads from 4 C and leave at room temperature for at least 15 minutes before use. It is important to ensure that they have completely reached room temperature before use. Cold beads will result in reduced recovery. Fully resuspend beads by inverting and tapping before adding to sample. Note that we recommend using 1.4 volumes (28 µl) of RNAClean XP beads during the post-second strand purification. This bead ratio has been optimized for use in this protocol. It is important to note that this is different than the standard Agencourt protocol and other NuGEN protocols that employ Agencourt beads. It is critical to let the beads separate on the magnet for a full five minutes. Removing binding buffer before the beads have completely separated will impact cdna yields. After the binding step has been completed, it is important to minimize bead loss when removing the binding buffer. With the samples placed on the magnet, remove only 40 µl of the binding buffer from each sample. Some liquid will remain at the bottom of the tube but this will minimize bead loss. Any significant loss of beads bound to the magnet during the ethanol washes will impact cdna yields, so make sure the beads are not lost with the wash. Ensure that the ethanol wash solution is freshly prepared from fresh ethanol stocks as lower percent ethanol mixes will reduce recovery. During the ethanol washes, keep the samples on the magnet. The beads should not be allowed to disperse; the magnet will keep the beads on the walls of the sample wells or tubes in a small ring. It is critical that all residual ethanol be removed prior to continuing with the SPIA amplification. Therefore, when removing the final ethanol wash, first remove most of the ethanol, then allow the excess to collect at the bottom of the tube before removing the remaining ethanol. This ensures beads dry completely during the recommended bead air-drying time (15 20 minutes). After drying the beads for at least minutes, inspect each tube carefully and make certain that all ethanol has evaporated before proceeding with the amplification step. 12 Ovation One-Direct System

16 IV. Protocol It is strongly recommended that strip tubes or partial plates are firmly placed when used with the magnet plate. We strongly discourage the use of individual reaction tubes as they can shift on the magnet plates, potentially resulting in poor recovery. D. Programming the Thermal Cycler Use a thermal cycler with a heat block designed for 0.2 ml tubes, equipped with a heated lid, and with a capacity of 100 µl reaction volume. Prepare the programs shown in Table 6, following the operating instructions provided by the manufacturer. For thermal cyclers with an adjustable heated lid, set the lid temperature at 100 C. For thermal cyclers with a fixed temperature heated lid (e.g. ABI GeneAmp PCR 9600 and 9700 models) use the default settings (typically 100 to 105 C). Table 6. Thermal Cycler Programming FIRST STRAND cdna SYNTHESIS Program 1 First Strand Primer Annealing Program 2 First Strand Synthesis 65 C 2 min, hold at 4 C 4 C 2 min, 25 C 30 min, 48 C 30 min, 95 C 5 min, hold at 4 C SECOND STRAND cdna SYNTHESIS Program 3 Second Strand Synthesis 4 C 1 min, 25 C 10 min, 50 C 30 min, 80 C 20 min, hold at 4 C SPIA AMPLIFICATION Program 4 SPIA Amplification 4 C 1 min, 47 C 90 min, 95 C 5 min, hold at 4 C POST-SPIA AMPLIFICATION Program 5 Post-SPIA Primer Annealing Program 6 Post-SPIA Modification 95 C 5 min, hold at 4 C 4 C 1 min, 30 C 10 min, 42 C 60 min, 75 C 10 min, hold at 4 C 13 Ovation One-Direct System

17 IV. Protocol E. First Strand cdna Synthesis Important Note: Carry out steps E (First-strand Synthesis) through H (SPIA Amplification) in a pre-amplification workspace using dedicated preamplification consumables and equipment. Wipe all surfaces, equipment and instrumentation with a DNA decontaminant solution such as DNA-OFF (MP Biomedicals, Cat# QD0500) to avoid the potential introduction of previously amplified cdna into new amplifications. For more information on our recommendations for workflow compartmentalization and routine lab cleanup please refer to Appendix F of this user guide. If you have any questions on this important topic, please contact NuGEN Technical Services (techserv@nugen.com, or ). Start here if using cell lysates prepared with the Direct Lysis Buffer: 1. Obtain and count cells to be processed. 2. Deliver cells to at least 2 µl of Direct Lysis Buffer. More Direct Lysis Buffer can be used if larger numbers of cells are being processed. (Note: the maximum number of cells that can be lysed in a given volume of Direct Lysis Buffer is dependent on the amount of total RNA present in the particular cell type used, which can vary widely. As a general guideline, strive for no more than 5 20 cells per 2 µl of Direct Lysis Buffer. Some experimentation may be necessary to determine the optimum cell numbers and working limits for a given cell type.) 3. Vortex for 1 minute to lyse cells. 4. Place cell lysate(s) on ice. 5. Obtain First Strand Buffer Mix (blue: A2), First Strand Enzyme Mix (blue: A3), and Nuclease-free Water (green: D1) from the components stored at 20 C and the First Strand Primer Mix (blue: A1) stored at 80 C. 6. Flick to mix, then spin down contents of A3 for 2 seconds and place on ice. 7. Thaw the other reagents at room temperature. Mix by vortexing for 2 seconds then spin for 2 seconds and place on ice. 8. Add 2 µl of the cell lysate and 3 µl Nuclease-free Water (D1) to a 0.2 ml PCR tube. Do not add more than 2 µl of lysate to a reaction as the Direct Lysis Buffer can inhibit amplification at higher levels. 9. Add 2 µl of A1 to each reaction tube. Mix well by pipetting. 10. Cap and spin tube(s) for 2 seconds and return tubes to ice. 14 Ovation One-Direct System

18 IV. Protocol 11. Place tubes in a pre-warmed thermal cycler programmed to run Program 1 (First Strand Primer Annealing; see Table 6): 65 C 2 min, hold at 4 C 12. Remove tubes from the thermal cycler and place on ice. 13. Once Primer Annealing (Step 11) is complete, prepare a master mix by combining A2 and A3 in a 0.5 ml capped tube, according to the volumes shown in Table 7 or other user-defined settings that produce fragmented DNA with an average size of bases: Table 7. First Strand Master Mix for use with cell lysate samples (volumes listed are for a single reaction) Mix by pipetting and spin down the master mix briefly. Immediately place on ice. FIRST STRAND BUFFER MIX (BLUE: A2 ver 5) FIRST STRAND ENZYME MIX (BLUE: A3 ver 3) 2.5 µl 0.5 µl 14. Proceed with step #10 below. Start here if using purified total RNA as input: 1. Obtain First Strand Buffer Mix (blue: A2), First Strand Enzyme Mix (blue: A3), and Nuclease-free Water (green: D1) from the components stored at 20 C and the First Strand Primer Mix (blue: A1) stored at 80 C. 2. Flick to mix, then spin down contents of A3 for 2 seconds and place on ice. 3. Thaw the other reagents at room temperature. Mix by vortexing for 2 seconds then spin for 2 seconds and place on ice. Leave water, D1 at room temperature. 4. Add 5 µl of purified total RNA sample (1 500 pg) to a 0.2 ml PCR tube. 5. Add 2 µl of A1 to each reaction tube. Mix well by pipetting. 6. Cap and spin tubes for 2 seconds and return to ice. 7. Place tubes in a pre-warmed thermal cycler programmed to run Program 1 (First Strand Primer Annealing; see Table 6): 65 C 2 min, hold at 4 C 8. Remove tubes from the thermal cycler and place tubes on ice. 9. Once Primer Annealing (Step 7) is complete, prepare a master mix by combining A2, A3, and D1 in a 0.5 ml capped tube according to the volumes shown in Table Ovation One-Direct System

19 IV. Protocol Table 8. First Strand Master Mix for use with purified total RNA samples (volumes listed are for a single reaction). In order to ensure accurate measurement of the A3 reagent, do not make this mix for fewer than 4 reactions. Mix by pipetting and spin down the master mix briefly. Immediately place on ice. FIRST STRAND BUFFER MIX (BLUE: A2 ver 5) FIRST STRAND ENZYME MIX (BLUE: A3 ver 3) WATER (GREEN: D1) 2.5 µl 0.25 µl 0.25 µl Note: continue here if using the direct cell lysis protocol 10. Add 3 µl of the First Strand master mix to each tube. 11. Mix well by pipetting, spin for 2 seconds. 12. Place tubes in a pre-cooled thermal cycler programmed to run Program 2 (First Strand Synthesis; see Table 6): 4 C 2 min, 25 C 30 min, 48 C 30 min, 95 C 5 min, hold at 4 C 13. Remove tubes from the thermal cycler, spin for 2 seconds to collect condensation, then place on ice. 14. Continue immediately with second strand cdna synthesis. F. Second Strand cdna Synthesis The purification beads should be removed from 4 C storage and left on the bench top to reach room temperature well before the start of purification. 1. Remove the Agencourt RNAClean XP purification beads from 4 C storage and place on the bench top to reach room temperature for use in the next step. 2. Obtain the Second Strand Buffer Mix (yellow: B1) and the Second Strand Enzyme Mix (yellow: B2), from 20 C storage. 3. Flick B2 to mix, then spin down contents for 2 seconds and place on ice. 4. Thaw reagent B1 at room temperature, mix by vortexing, spin and place on ice. 5. Make a master mix by combining B1 and B2 in a 0.5 ml capped tube, according to the volumes shown in Table 9. Table 9. Second Strand Master Mix (volumes listed are for a single reaction) Mix by pipetting and spin down the master mix briefly. Immediately place on ice. SECOND STRAND BUFFER MIX (YELLOW: B1 ver 3) SECOND STRAND ENZYME MIX (YELLOW: B2 ver 2) 9.6 µl 0.4 µl 6. Add 10 µl of the Second Strand Master Mix to each First Strand reaction tube. 16 Ovation One-Direct System

20 IV. Protocol 7. Mix by pipetting 5 times, spin and place on ice. 8. Place the tubes in a pre-cooled thermal cycler programmed to run Program 3 (Second Strand Synthesis; see Table 6): 4 C 1 min, 25 C 10 min, 50 C 30 min, 80 C 20 min, hold at 4 C 9. Remove the tubes from the thermal cycler, spin to collect condensation then place on ice. 10. (Optional) A 2 µl aliquot of the Second Strand cdna may be removed at this point for analytical purposes. 11. Continue immediately with the Purification of cdna protocol. G. Purification of cdna 1. Ensure the Agencourt RNAClean XP beads have completely reached room temperature before proceeding. Best results are obtained by using fresh 70% ethanol in the wash step. 2. Prepare a 70% ethanol wash solution. It is critical that this solution be prepared fresh on the same day of the experiment from a recently opened stock container. Measure both the ethanol and the water components carefully prior to mixing. Failure to do so can result in a higher than anticipated aqueous content, which may reduce amplification yield. 3. Resuspend the beads by inverting and tapping the tube. Ensure that the beads are fully resuspended before adding to the sample. After resuspending, do not spin the beads. A large excess of beads is provided; therefore, it is not necessary to recover any trapped in the cap. 4. At room temperature, add 28 µl (1.4 volumes) of the bead suspension to each reaction and mix by pipetting 10 times. 5. Incubate at room temperature for 20 minutes. 6. Transfer the tubes to the magnet and let stand 5 minutes to completely clear the solution of beads. Minimize bead loss by leaving a residual volume of binding buffer after completion of the binding step. Best results can be obtained by using fresh 70% ethanol in the wash step. 7. Keeping the tubes on the magnet, carefully remove only 40 µl of the binding buffer and discard it. Leaving some of the volume behind minimizes bead loss at this step. 8. With the tubes still on the magnet, add 200 µl of freshly prepared 70% ethanol and allow to stand for 30 seconds. Note: The beads should not disperse; instead, they will stay on the walls of the tubes. Significant loss of beads at this stage will impact cdna yields, so ensure beads are not removed with the binding buffer or the washes. 9. Remove the 70% ethanol wash using a pipette. 10. Repeat the wash two more times. 17 Ovation One-Direct System

21 IV. Protocol Note: With the final wash, it is critical to remove as much of the ethanol as possible. Use at least 2 pipetting steps and allow excess ethanol to collect at the bottom of the tubes after removing most of the ethanol in the first pipetting step. 11. Air-dry the beads on the magnet for minutes. Inspect each tube carefully to ensure that all the ethanol has evaporated. It is critical that all residual ethanol be removed prior to continuing with SPIA amplification. 12. Continue immediately with the SPIA Amplification protocol with the cdna still bound to the dry beads. H. SPIA Amplification 1. Obtain the SPIA Buffer Mix (red: C2), and SPIA Enzyme Mix (red: C3), stored at 20 C and the SPIA Primer Mix (red: C1) stored at 80 C. 2. Thaw reagents C1 and C2 at room temperature, mix by vortexing, spin and place on ice. 3. Thaw C3 on ice and mix the contents by inverting gently 5 times. Ensure the enzyme is well mixed without introducing bubbles, spin and place on ice. Use SPIA Master Mix immediately after preparation. 4. Make a master mix by sequentially combining C2, C1 and C3 in an appropriately sized, capped tube according to the volumes shown in Table 10. Note: Make sure the addition of C3 is at the last moment. Table 10. SPIA Master Mix (volumes listed are for a single reaction) Mix by pipetting and spin down the master mix briefly. Immediately place on ice. SPIA BUFFER MIX (RED: C2 ver 5) SPIA PRIMER MIX (RED: C1 ver 7) SPIA ENZYME MIX (RED: C3 ver 5) 75 µl 37.5 µl 37.5 µl 5. Add 150 µl of the SPIA Master Mix to each tube containing the double-stranded cdna bound to the dried beads. Use a pipette set to 75 µl and mix thoroughly by pipetting at least 8 10 times. Attempt to get the majority of the beads in suspension and remove most of the beads from the tube walls. Note: The beads may not form a perfectly uniform suspension, but this will not affect the reaction. The addition of SPIA master mix will elute the cdna from the beads. 6. Transfer one half of the reaction volume (75 µl) to a second tube. 7. Place the tubes in a pre-cooled thermal cycler programmed to run Program 4 (SPIA Amplification, see Table 6): 4 C 1 min, 47 C 90 min, 95 C 5 min, hold at 4 C 8. Remove the tubes from thermal cycler, spin to collect condensation and place on ice. Do not re-open the tubes in the pre-amplification workspace. 18 Ovation One-Direct System

22 IV. Protocol Important Note: At this point the tubes should be removed from the pre-amplification workspace. Carry out all remaining steps in a postamplification workspace using dedicated post-amplification consumables and equipment. Take care to avoid the introduction of previously amplified cdna into your pre-amplification workspace. For more information on our recommendations for workflow compartmentalization and routine lab cleanup, please refer to Appendix F of this user guide. If you have any questions on this important topic, please contact NuGEN Technical Services (techserv@nugen.com, or ). 9. Continue immediately with the Post-SPIA Modification or store SPIA cdna at 20 C. I. Post-SPIA Modification 1. Transfer tubes or plate containing the half-reactions to the SPRIPlate Super Magnet Plate and let stand 5 minutes to completely clear the solution of beads. 2. Carefully remove 70 µl of the cleared supernatant from each half-reaction and transfer to a fresh tube or plate and place on ice. 3. (Optional) A 2 µl aliquot of the SPIA cdna may be removed from the volume remaining in the original tube or plate for analytical purposes. 4. The beads may now be discarded. 5. Obtain the Primer Mix (Violet: E1), Buffer Mix (Violet: E2) and Enzyme Mix (Violet: E3) from the product box stored at 20 C. 6. Thaw E1 and E2 at room temperature and mix by vortexing, spin and place on ice. 7. Mix E3 by inverting the tube three times. Spin, then place on ice. 8. Add 10 µl E1 Primer Mix to each of the two half-reactions. Mix well by pipetting up and down 8 10 times. 9. Close the tubes or seal the plate tightly, spin down briefly and place the tubes or plate in a thermal cycler programmed to run Program 5 (Post-SPIA Primer Annealing,see Table 6): 95 C 5 min, hold at 4 C 10. Remove tubes from the thermal cycler, spin for 2 seconds to collect condensation and place on ice. 11. Make Post-SPIA Modification Master Mix as shown in Table Ovation One-Direct System

23 IV. Protocol Table 11. Post-SPIA Modification Master Mix (volumes listed are for a single reaction) Mix by pipetting and spin down the master mix briefly. Immediately place on ice. BUFFER MIX (VIOLET: E2 ver 2) ENZYME MIX (VIOLET: E3 ver 1) 20 µl 20 µl 12. Add 20 µl of the Post-SPIA Modification Master Mix to each half-reaction. 13. Mix well by pipetting up and down 8 10 times. 14. Seal tubes or plate and place in a pre-cooled thermal cycler programmed to run Program 6 (Post-SPIA Modification, see Table 6): 4 C 1 min, 30 C 10 min, 42 C 60 min, 75 C 10 min, hold at 4 C 15. Remove tubes or plate from the thermal cycler, spin for 2 seconds to collect condensation. Recombine half reactions into a singe tube and place on ice. 16. Proceed immediately to Purification of Amplified cdna or store SPIA cdna at 20 C. J. Purification of Amplified cdna Protocol Purification of the amplified cdna is required if it is intended for use with an Encore Biotin Module or other supported labeling protocol for use in microarray experiments. We strongly recommend that the amplified SPIA cdna product be purified prior to qpcr analysis. The recommended method is the QIAGEN MinElute Spin Column, described below. Note that the protocol below differs slightly from the manufacturer s protocol. You must follow the procedures outlined in this user guide for the use of these columns with the Ovation One-Direct System. For alternative purification methods see Appendix C. 100% ethanol must be added to the QIAGEN Buffer PE upon first use. Failure to do so will result in low amplification yields. QIAGEN MinElute Spin Column (instructions for a single full reaction, 2 columns are required per reaction) Ensure that 100% ethanol has been added to Buffer PE as described in the QIAGEN MinElute Handbook. 1. Failure to add ethanol to this buffer will result in low amplification yield. 2. Prepare an 80% ethanol wash solution. It is critical that this solution be prepared fresh on the day of the experiment from a recently opened stock container. Measure both the ethanol and the water components carefully prior to mixing. Failure to do so can result in a higher than anticipated aqueous content which may reduce amplification yield. 3. Add 600 µl of Buffer ERC to a labeled 1.5 ml tube for each amplification reaction. 20 Ovation One-Direct System

24 IV. Protocol 4. Transfer each full reaction (200 µl) into a tube containing the Buffer ERC. 5. Vortex for 5 seconds and spin down briefly. 6. Obtain and label two QIAGEN MinElute Spin Columns for each amplification reaction and place them into collection tubes. 7. Load 400 µl (one-half) of each reaction/buffer mix onto each of the two labeled QIAGEN MinElute Spin Columns. 8. Centrifuge columns in the collection tube for 1 minute at full speed in a microcentrifuge. 9. Discard flow-through and replace the QIAGEN MinElute Spin Column in the same collection tube. 10. Wash sample by adding 500 µl of Buffer PE (prepared according to manufacturer s recommendations). Centrifuge column in the collection tube for 1 minute at full speed. Discard flow-through. Best results are obtained by using fresh 80% ethanol in the wash step. Lower percent ethanol mixes will reduce recovery. 11. Add 500 µl of the room temperature 80% ethanol prepared in Step 1 above. Note: Use fresh 80% ethanol. 12. Centrifuge column in the collection tube for 1 minute at full speed. Discard flow-through. 13. Place the column back in the same collection tube and spin for an additional 2 minutes at full speed. Important: Residual ethanol from the wash buffers will not be completely removed unless the flow-through is discarded before this additional centrifugation. 14. Blot the column tip onto a filter paper to remove any residual wash buffer from the tip of the column. Note: Blot column tip prior to transferring it to a new tube to prevent any wash buffer transferring to the eluted sample. 15. Place the MinElute Column in a clean, labeled 1.5 ml microcentrifuge tube. Use nuclease-free water at room temperature to elute sample 16. Add 16 µl of room temperature Nuclease-free Water (green: D1) from the kit to the center of each column. Do not use cold water! Important: Ensure that the water is dispensed directly onto the membrane for complete elution of bound cdna. 17. Let columns stand for 1 minute at room temperature. 18. Centrifuge column and microcentrifuge tube for 1 minute at full speed. 19. Pool eluates from each half-reaction and measure the volume recovered. There should be approximately µl of purified cdna. 20. Mix sample by vortexing, then spin briefly. 21 Ovation One-Direct System

25 IV. Protocol 21. Proceed to Measuring cdna Product Yield and Purity or store purified cdna at 20 C. K. Measuring cdna Product Yield and Purity 1. Mix the sample by brief vortexing and spinning prior to checking the concentration. 2. Measure the absorbance at 260, 280 and 320 nm. You may need to make a 1:20 dilution of the cdna in water prior to measuring the absorbance. 3. Purity: Subtract the A320 value from both A260 and A280 values. The adjusted (A260 A320) / (A280 A320) ratio should be > Yield: Assume 1 A260 unit = 33 µg/ml. To calculate: (A260 A320 of diluted sample) X (dilution factor) X 33 (concentration in µg/ml of a 1 A260 unit solution) X 0.03 (final volume in ml) = total yield in micrograms Note: Alternatively, you may measure the concentration and purity of cdna with a Nanodrop by setting 1 A260 unit = 33 µg/ml as the constant. 5. The purifed cdna may be stored at 20 C. 22 Ovation One-Direct System

26 V. Technical Support For help with any of our products, please contact NuGEN Technical Support at (direct) or , option 2 (toll-free, US only). You may also send faxes to (toll-free) or techserv@nugen.com. In Europe contact NuGEN at +31(0) (Phone) or +31(0) (Fax) or europe@nugen.com. In all other locations, contact your NuGEN distributor's Technical Support team. 23 Ovation One-Direct System

27 VI. Appendix A. Considerations for Microarray Analysis To optimize array performance and achieve the best signal intensity on arrays when using these extremely small samples, it is recommended that you allow the array hybridization to proceed for a longer period of time (~40 hours for GeneChip arrays). You may also increase the final concentration of fragmented, labeled target in the hybridization cocktail up to 27 ng/µl. B. Performing Quantitative PCR on Amplified cdna It is recommended that the amplified cdna generated from the Ovation One-Direct System should be purified prior to use in real time quantitative PCR reactions. Since different amplified cdna samples may be variable in concentration, the purified products can be quantitated and mass normalized to ensure the cdna inputs to qpcr are equal for all samples. Purified amplified cdna produced with the kit has been successfully used as template for qpcr systems including TaqMan and SYBR Green. Note that RT-PCR master mixes containing the enzyme Uracil N-Glycosylase (UNG) are not compatible with the Ovation One-Direct System. NuGEN can recommend the following reagents for qpcr: TaqMan: ABsolute qpcr Mix plus ROX (ABgene, Cat. #AB-1136/B), Fast Universal PCR Master Mix 2x (Applied Biosystems, Cat. # ) SYBR: QuantiTect SYBR Green PCR Kit (QIAGEN, Cat. #204143), iq SYBR Green Supermix (BioRad, Cat. # ), FastStart SYBR Green Master (ROX) (Roche, Cat. # ) 1. Recommendations to Achieve Optimal Results A. Dilute the Amplified Product After purification and quantitation of amplified cdna, it can be diluted to an appropriate concentration for qpcr reaction. It will be necessary to empirically determine the appropriate input of amplified cdna for use in qpcr reactions. Higher cdna inputs may be required for qpcr from amplifications from very low numbers of cells or RNA input. Depending on the abundance of the transcripts of interest you may wish to use more or less cdna. B. Primer Design We recommend using primers and probes designed with amplicon sizes as small as possible. Primers may be designed at any position along a transcript since the Ovation amplification covers the whole transcriptome. C. Alternative Purification Protocols for Amplified cdna The recommended SPIA cdna purification method is using the QIAGEN MinElute Spin Column. Other alternative methods are listed below. 24 Ovation One-Direct System

28 VI. Appendix QIAGEN QIAquick PCR Purification Kit (instructions for a single reaction) Important Notes: Prepare an 80% ethanol wash solution. It is critical that this solution be prepared fresh on the same day of the experiment from a recently opened stock container. Measure both the ethanol and the water components carefully prior to mixing. Failure to do so can result in a higher than anticipated aqueous content which may reduce amplification yield. All centrifugation steps are carried out at 17,900 X g (13,000 RPM) in a conventional tabletop microcentrifuge at room temperature. It is not necessary to add the ph Indicator I from the QIAGEN kit to Buffer PB for this protocol. 1. Into a clean, labeled 1.5 ml microcentrifuge tube, add 1000 μl of Buffer PB from the QIAGEN kit. 2. Add the 200 µl of amplified cdna product to the tube. 3. Vortex for 5 seconds, then spin briefly. 4. Obtain and label a QIAquick spin column and place it into a collection tube. 5. Load 600 µl of sample onto the column. 6. Centrifuge for 1 minute at 17,900 X g in a microcentrifuge. 7. Discard the flow-through and replace the column in the same collection tube. 8. Load remaining 600 µl onto the same column. Centrifuge column in collection tube for 1 minute at 17,900 X g. Discard flow-through and place the column back in the same collection tube. Best results are obtained by using fresh 80% ethanol in wash steps. Lower percent ethanol mixes will reduce recovery. 9. Add 700 μl of 80% ethanol to the column. 10. Centrifuge for 1 minute at 17,900 X g. 11. Discard the flow-through and replace the column in the same collection tube. 12. Repeat steps 9 through 11 once. 13. Centrifuge the column for an additional 1 minute at 17,900 X g to remove all residual ethanol. Note: Residual ethanol from the wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. 14. Discard the flow-through along with the collection tube. Blot the column tip onto clean, absorbant paper to remove any residual wash buffer from the tip of the column. Note: Blotting the column tip prior to transferring it to a new tube is necessary to prevent any wash buffer transferring to the eluted sample. 15. Place the column into a clean, labeled 2.0 ml microcentrifuge tube. 25 Ovation One-Direct System