TaqMan Advanced mirna Assays

Transcription

1 TaqMan Advanced mirna Assays USER GUIDE TaqMan OpenArray Plates for use with: TaqMan Advanced mirna cdna Synthesis Kit Publication Number MAN Revision A.0 For Research Use Only. Not for use in diagnostic procedures.

2 Manufacturer: Life Technologies Corporation 6055 Sunol Blvd Pleasanton, CA The information in this guide is subject to change without notice. DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT. Revision history: Pub. No. MAN Revision Date Description A.0 13 September 2017 New document. Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses. Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license Thermo Fisher Scientific Inc. All rights reserved.

3 Contents CHAPTER 1 Product information... 5 Product description... 5 TaqMan OpenArray Plate overview... 6 Contents and storage... 7 Required materials... 7 Endogenous and exogenous controls... 9 Workflow CHAPTER 2 Prepare cdna templates Procedural guidelines Guidelines for RNA input Guidelines for preparing cdna templates Perform the poly(a) tailing reaction Perform the adaptor ligation reaction Perform the reverse transcription (RT) reaction Perform the mir-amp reaction CHAPTER 3 Prepare and run TaqMan Advanced mirna Assays with OpenArray plates Generate OpenArray plate layouts in the QuantStudio 12K Flex Software Set up the real-time PCR reactions in an OpenArray 384-well Sample Plate Set up the AccuFill instrument Transfer reactions to the OpenArray plate (AccuFill instrument) Seal the OpenArray plate Run the OpenArray plate(s) on the QuantStudio 12K Flex instrument Check the QC images TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates) 3

4 Contents CHAPTER 4 Export and review TaqMan Advanced mirna Assay data Export data Prepare exported data for analysis Review results Fields for Pivot Tables APPENDIX A Troubleshooting Troubleshoot with cycling and imaging run images (QC images) AccuFill instrument plate loading errors OpenArray plate assembly and handling errors APPENDIX B Supplemental information Endogenous and exogenous controls Endogenous controls Exogenous controls cdna template preparation TaqMan Advanced mirna Assays chemistry overview TaqMan MGB probes About the 5' nuclease assay Best practices for PCR and RT-PCR experiments Good laboratory practices for PCR and RT-PCR Use UNG to prevent false-positive amplification Detect fluorescent contaminants APPENDIX C Safety Chemical safety Biological hazard safety Documentation and support Related documentation Customer and technical support Limited product warranty References TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates)

5 1 Product information Product description... 5 TaqMan OpenArray Plate overview... 6 Contents and storage... 7 Required materials... 7 Workflow Product description TaqMan Advanced mirna Assays are pre-formulated primer and probe sets that are designed for analysis of microrna (mirna) expression levels using the QuantStudio 12K Flex Real-Time PCR System and the OpenArray AccuFill System. The assays can detect and quantify the mature form of the mirna from 5 10 ng of total RNA from tissue, or 2 µl of total RNA from serum or plasma. For more information about PCR detection with TaqMan Advanced mirna Assays, see page 33. The TaqMan Advanced mirna cdna Synthesis Kit (Cat. No. A28007; sold separately) is required for preparing the cdna template that is used with the TaqMan Advanced mirna Assays. The kit enables the analysis of: Multiple mirnas from a single amplified sample. Samples that are limited in quantity, including serum, plasma, or other biological fluids. This document describes procedures to prepare cdna templates from mirna followed by PCR amplification of the cdna template and subsequent data analysis. The procedures in this document are for use with TaqMan Advanced mirna Assays configured on predefined TaqMan OpenArray Plates (see Contents and storage on page 7). In the first stage of the workflow, mature mirnas from total RNA are modified by 1) extending the 3' end of the mature transcript through poly(a) addition, then 2) lengthening the 5 end by adaptor ligation. The modified mirnas then undergo universal reverse transcription followed by amplification to increase uniformly the amount of cdna for all mirnas (mir-amp reaction). For more information about cdna synthesis of templates for TaqMan Advanced mirna Assays, see page 32. TaqMan Advanced mirna Assays of interest are then used for quantification of mirna expression levels by qpcr analysis. Predesigned TaqMan Advanced mirna Assays are available for most human mirnas in mirbase (the mirna sequence repository). For a current list of assays, go to thermofisher.com/advancedmirna. TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates) 5

6 1 Chapter 1 Product information TaqMan OpenArray Plate overview Note: TaqMan Advanced mirna Assays are for analysis of mature mirna only. For analysis of sirna, or other small RNAs that are fewer than 200 bases in length, go to thermofisher.com/taqmanmirna. TaqMan OpenArray Plate overview TaqMan OpenArray Plates enable high-throughput screening experiments that use a small amount of sample and reagent. Each plate is the size of a microscope slide and contains 48 subarrays. Each subarray has 64 through-holes, for a total of 3,072 through-holes on the plate. Each TaqMan OpenArray Plate is equivalent to eight 384-well plates. Assays are pre-loaded into the through-holes during the manufacturing process. Each through-hole is 300 µm in diameter and 300 µm deep. Through-holes are treated with hydrophilic coatings and the plate surface is treated with hydrophobic coatings so that 33 nl of reagent is retained in each through-hole by surface tension. To run a TaqMan OpenArray Plate, samples are mixed with master mix, loaded into the TaqMan OpenArray Plate through-holes with the automated OpenArray AccuFill System, then cycled and imaged with the QuantStudio 12K Flex Real-Time PCR System Subarray 2 Hydrophobic coating on plate surface 3 Hydrophilic coating in through-hole 4 Through-hole 6 TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates)

7 Chapter 1 Product information Contents and storage 1 Contents and storage Preplated and predefined TaqMan OpenArray Plates configured with TaqMan Advanced mirna Assays are manufactured and stocked in advance. For the template file (EDT), visit thermofisher.com/taqmanfiles. Table 1 TaqMan Advanced mirna Assays (TaqMan OpenArray Plate format) Item Cat. No. Amount Storage TaqMan OpenArray Human Advanced mirna Panel A plate 25 C to 15 C Required materials Unless otherwise indicated, all materials are available through thermofisher.com. MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier. Table 2 Recommended RNA isolation kits Sample type Kit Cat. No. Tissue samples mirvana mirna Isolation Kit, with phenol AM1560 mirvana mirna Isolation Kit, without phenol MagMAX mirvana Total RNA Isolation Kit mirvana PARIS RNA and Native Protein Purification Kit AM1561 A27828 AM1556 Serum / Plasma samples Total Exosome RNA and Protein Isolation Kit MagMAX mirvana Total RNA Isolation Kit A27828 mirvana PARIS RNA and Native Protein Purification Kit AM1556 Cell samples TaqMan MicroRNA Cells-to-C T Kit FFPE samples RecoverAll Total Nucleic Acid Isolation Kit AM1975 Table 3 TaqMan Advanced mirna cdna Synthesis Kit (Cat. No. A28007): 50 reactions Component Storage 10X Poly(A) Buffer ATP, 10 mm Poly(A) Enzyme, 5 U/µL 5X DNA Ligase Buffer 20 C RNA Ligase, 10 U/µL 50% PEG 8000 TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates) 7

8 1 Chapter 1 Product information Required materials Component Storage 25X Ligation Adaptor 10X RT Enzyme Mix 5X RT Buffer 20X Universal RT Primer 20 C dntp Mix, 100 mm 20X mir-amp Primer Mix 2X mir-amp Master Mix 2 4 C Table 4 Other materials and equipment required for the workflow Item Source Real-time PCR instrument: QuantStudio 12K Flex Real-Time PCR System with the OpenArray AccuFill System Contact your local sales office Software ImageJ (To view quality control images) imagej.nih.gov/ig Equipment QuantStudio 12K Flex OpenArray Plate Press 2.0 Thermal cycler, one of the following (or equivalent): GeneAmp PCR System 9700 Veriti Thermal Cycler Centrifuge, capable of spinning sample plates at 1,000 rpm Microcentrifuge Vortex mixer (Optional) Eppendorf MixMate (shaker) Pipettes A24945 Contact your local sales office MLS MLS MLS Fisher Scientific MLS Tubes, plates, and other consumables Plastics consumables Adhesive PCR Plate Foils thermofisher.com/ plastics AB0626 OpenArray AccuFill System Tips QuantStudio 12K Flex OpenArray Accessories Kit [1] TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates)

9 Chapter 1 Product information Required materials 1 Item OpenArray 384-well Sample Plates Forceps Aerosol-resistant barrier pipette tips Disposable gloves Source (clear) (black) MLS MLS MLS Reagents Nuclease-Free Water Tris EDTA Buffer (TE, ph 8.0) AM9930 AM9849 TaqMan OpenArray Real-Time PCR Master Mix [1] Contains items to assemble 10 OpenArray plates (12 lids and plugs, 12 immersion fluid syringes, and 12 carriers. Endogenous and exogenous controls For information about using endogenous or exogenous controls with TaqMan Advanced mirna Assays, see page 31. See A technical guide to identifying mirna normalizers using TaqMan Advanced mirna Assays White Paper (Pub. No. COL ) for available TaqMan Advanced mirna Assays that target mirnas with relatively constant expression levels across many different sample types (available at thermofisher.com/advancedmirna; in the Documentation section). Table 5 Endogenous control Assay name [1] Assay ID Target Sequence hsa-mir-16-5p _mir 5 -UAGCAGCACGUAAAUAUUGGCG-3 [1] TaqMan Advanced mirna Assays do not detect snrnas or snornas. Do not use snrnas and snornas as endogenous controls for these assays. Table 6 Assay options for exogenous controls (for human samples) Assay Name Assay ID Target Sequence [1] ath-mir159a _mir 5 -UUUGGAUUGAAGGGAGCUCUA-3 cel-mir-39-3p _mir 5 -UCACCGGGUGUAAAUCAGCUUG-3 [1] Oligonucleotides for exogenous controls must be 5'-phosphorylated. TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates) 9

10 1 Chapter 1 Product information Workflow Workflow Prepare cdna templates Input RNA sample Perform the poly(a) tailing reaction (page 12) Perform the adaptor ligation reaction (page 13) Perform the reverse transcription (RT) reaction (page 14) Perform the mir-amp reaction (page 15) Prepare and run TaqMan Advanced mirna Assays with OpenArray plates Generate OpenArray plate layouts in the QuantStudio 12K Flex Software (page 16) Set up the real-time PCR reactions in an OpenArray 384-well Sample Plate (page 17) Set up the AccuFill instrument (page 19) Transfer reactions to the OpenArray plate (AccuFill instrument) (page 20) Seal the OpenArray plate (page 21) Run the OpenArray plate(s) on the QuantStudio 12K Flex instrument (page 22) Export and review TaqMan Advanced mirna Assay data Export data (page 24) Prepare exported data for analysis (page 24) Review results (page 25) 10 TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates)

11 2 Prepare cdna templates Procedural guidelines Perform the poly(a) tailing reaction Perform the adaptor ligation reaction Perform the reverse transcription (RT) reaction Perform the mir-amp reaction Procedural guidelines Guidelines for RNA input Prepare samples using a total RNA isolation method that preserves small RNAs. See Required materials on page 7 for recommended RNA isolation kits. For tissue samples: Use 5 10 ng of total RNA per reaction. Note: Sample concentration before adding to reactions should be 5 ng/µl. For blood, serum, or plasma samples: Use 2 µl of sample eluent (from the sample isolation procedure) per reaction. If RNA can be quantified, use 5 10 ng of total RNA per reaction. For optimal reverse transcription, input RNA should be: Free of inhibitors of reverse transcription (RT) and PCR Dissolved in PCR-compatible buffer Free of RNase activity Nondenatured total RNA (not applicable for double-stranded templates) IMPORTANT! Do not denature the total RNA. Guidelines for preparing cdna templates Follow best practices when working with RNA samples (see Best practices for PCR and RT-PCR experiments on page 35). Calculate the number of required reactions. Scale reaction components based on the single-reaction volumes, then include 10% overage. If using strip tubes, change to a new strip cap after each step or incubation. See your instrument user guide for detailed instructions about using plates, tubes, or strip tubes. TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates) 11

12 2 Chapter 2 Prepare cdna templates Perform the poly(a) tailing reaction Perform the poly(a) tailing reaction 1. Thaw samples and cdna synthesis reagents on ice, gently vortex to thoroughly mix, then centrifuge briefly to spin down the contents and eliminate air bubbles. Note: Do not remove assays from storage until PCR setup. IMPORTANT! The 50% PEG 8000 reagent must be at room temperature for the adaptor ligation reaction (next section). 2. In a 1.5-mL microcentrifuge tube, prepare sufficient Poly(A) Reaction Mix for the required number of reactions according to the following table. Component 1 Rxn 4 Rxns [1] 10 Rxns [1] 10X Poly(A) Buffer 0.5 µl 2.2 µl 5.5 µl ATP 0.5 µl 2.2 µl 5.5 µl Poly(A) Enzyme 0.3 µl 1.3 µl 3.3 µl RNase-free water 1.7 µl 7.5 µl 18.7 µl Total Poly(A) Reaction Mix volume 3.0 µl 13.2 µl 33 µl [1] Volumes include 10% overage. 3. Vortex the Poly(A) Reaction Mix to thoroughly mix the contents, then centrifuge briefly to spin down the contents and eliminate air bubbles. 4. Add 2 µl of sample to each well of a reaction plate or each reaction tube. Note: (Optional) Before adding the sample to the reaction plate or tube, add RNase Inhibitor Protein to each sample to minimize the effects of RNase contamination. For detailed instructions, see the documentation provided by the RNase Inhibitor Protein manufacturer. 5. Add 3 µl of Poly(A) Reaction Mix to each well or tube. The total volume should be 5 µl per well or tube. 6. Seal the reaction plate or tubes, then vortex briefly to thoroughly mix the contents. 7. Centrifuge the reaction plate or tubes briefly to spin down the contents and eliminate air bubbles. 8. Place the reaction plate or tubes into a thermal cycler, then incubate using the following settings and standard cycling: Step Temperature Time Polyadenylation 37 C 45 minutes Stop reaction 65 C 10 minutes Hold 4 C Hold Proceed immediately to the adaptor ligation reaction (next section). 12 TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates)

13 Chapter 2 Prepare cdna templates Perform the adaptor ligation reaction 2 Perform the adaptor ligation reaction 1. In a 1.5-mL microcentrifuge tube, prepare sufficient Ligation Reaction Mix for the required number of reactions according to the following table. Component 1 Rxn 4 Rxns [1] 10 Rxns [1] 5X DNA Ligase Buffer 3 µl 13.2 µl 33 µl 50% PEG 8000 [2] 4.5 µl 19.8 µl 49.5 µl 25X Ligation Adaptor 0.6 µl 2.6 µl 6.6 µl RNA Ligase 1.5 µl 6.6 µl 16.5 µl RNase-free water 0.4 µl 1.8 µl 4.4 µl Total Ligation Reaction Mix volume 10 µl 44 µl 110 µl [1] Volumes include 10% overage. [2] 50% PEG 8000 is very viscous, follow the Important statement below to ensure accurate pipetting. IMPORTANT! For accurate pipetting of 50% PEG 8000: Use 50% PEG 8000 at room temperature. Aspirate and dispense solution slowly. a. Hold the pipette tip in the solution for ~10 seconds after slowly releasing the plunger during aspiration. This action allows the solution to be fully drawn into the pipette tip. b. Keep the plunger depressed for ~10 seconds to allow the solution to be fully dispensed into the Ligation Reaction Mix. 2. Vortex the Ligation Reaction Mix to thoroughly mix the contents, then centrifuge briefly to spin down the contents and eliminate air bubbles. 3. Transfer 10 µl of the Ligation Reaction Mix to each well of the reaction plate or each reaction tube containing the poly(a) tailing reaction product. The total volume should be 15 µl per well or tube. 4. Seal the reaction plate or tubes, then vortex briefly or shake (1,900 rpm for 1 minute with an Eppendorf MixMate ) to thoroughly mix the contents. IMPORTANT! If vortexing, watch for a swirling motion of the adaptor ligation reaction to ensure proper mixing. Proper mixing is necessary for efficient ligation. 5. Centrifuge the reaction plate or tubes briefly to spin down the contents. 6. Place the reaction plate or tubes into a thermal cycler, then incubate using the following settings and standard cycling: Step Temperature Time Ligation 16 C 60 minutes Hold 4 C Hold Proceed immediately to the reverse transcription (RT) reaction (next section). TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates) 13

14 2 Chapter 2 Prepare cdna templates Perform the reverse transcription (RT) reaction Perform the reverse transcription (RT) reaction 1. In a 1.5-mL microcentrifuge tube, prepare sufficient RT Reaction Mix for the required number of reactions according to the following table. Component 1 Rxn 4 Rxns [1] 10 Rxns [1] 5X RT Buffer 6 µl 26.4 µl 66 µl dntp Mix (25 mm each) 1.2 µl 5.3 µl 13.2 µl 20X Universal RT Primer 1.5 µl 6.6 µl 16.5 µl 10X RT Enzyme Mix 3 µl 13.2 µl 33 µl RNase-free water 3.3 µl 14.5 µl 36.3 µl Total RT Reaction Mix volume 15 µl 66 µl 165 µl [1] Volumes include 10% overage. 2. Vortex the RT Reaction Mix to thoroughly mix the contents, then centrifuge briefly to spin down the contents and eliminate air bubbles. 3. Transfer 15 µl of the RT Reaction Mix to each well of the reaction plate or each reaction tube containing the adaptor ligation reaction product. The total volume should be 30 µl per well or tube. 4. Seal the reaction plate or tubes, then vortex briefly to thoroughly mix the contents. 5. Centrifuge the reaction plate or tubes briefly to spin down the contents. 6. Place the reaction plate or tubes into a thermal cycler, then incubate using the following settings and standard cycling: Step Temperature Time Reverse transcription 42 C 15 minutes Stop reaction 85 C 5 minutes Hold 4 C Hold Proceed to the mir-amp reaction (next section) or store the RT reaction product at -20 C for up to 2 months. 14 TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates)

15 Chapter 2 Prepare cdna templates Perform the mir-amp reaction 2 Perform the mir-amp reaction 1. In a 1.5-mL microcentrifuge tube, prepare sufficient mir-amp Reaction Mix for the required number of reactions according to the following table. Component 1 Rxn 4 Rxns [1] 10 Rxns [1] 2X mir-amp Master Mix 25 µl 110 µl 275 µl 20X mir-amp Primer Mix 2.5 µl 11 µl 27.5 µl RNase-free water 17.5 µl 77 µl µl Total mir-amp Reaction Mix volume 45 µl 198 µl 495 µl [1] Volumes include 10% overage. 2. Vortex the mir-amp Reaction Mix to thoroughly mix the contents, then centrifuge briefly to spin down the contents and eliminate air bubbles. 3. Transfer 45 µl of the mir-amp Reaction Mix to each well of a new reaction plate or reaction tube. 4. Add 5 µl of the RT reaction product to each reaction well or each reaction tube. The total volume should be 50 µl per well or tube. 5. Seal the reaction plate or tubes, then vortex briefly to thoroughly mix the contents. 6. Centrifuge the reaction plate or tubes briefly to spin down the contents. 7. Place the reaction plate or tubes into a thermal cycler, then incubate using the following settings, maximum ramp speed, and standard cycling: Step Temperature Time Cycles Enzyme activation 95 C 5 minutes 1 Denature 95 C 3 seconds Anneal/Extend 60 C 30 seconds Stop reaction 99 C 10 minutes 1 Hold 4 C Hold 1 Proceed to performing the real-time PCR (next section) or store the undiluted mir-amp reaction product at 20 C for up to 2 months TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates) 15

16 3 Prepare and run TaqMan Advanced mirna Assays with OpenArray plates Generate OpenArray plate layouts in the QuantStudio 12K Flex Software Set up the real-time PCR reactions in an OpenArray 384-well Sample Plate Set up the AccuFill instrument Transfer reactions to the OpenArray plate (AccuFill instrument) Seal the OpenArray plate Run the OpenArray plate(s) on the QuantStudio 12K Flex instrument Check the QC images Generate OpenArray plate layouts in the QuantStudio 12K Flex Software Use the QuantStudio 12K Flex Software to map assays and samples onto the TaqMan OpenArray Plates. Three technical replicates of each reaction are recommended. Set up optimized folder locations and software preferences. Download the EDT file at thermofisher.com/taqmanfiles. The EDT file contains the assay layout on the TaqMan OpenArray Plate. 1. In Experiment menu, click Open, then select the EDT file. 2. Click Setup, then click Experiment Properties. 3. Enter the Experiment Name and the Barcode. 4. Click Define, then enter the sample names in the Samples screen. 5. Click Save as to save the assay information as an EDS file. 16 TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates)

17 Chapter 3 Prepare and run TaqMan Advanced mirna Assays with OpenArray plates Set up the real-time PCR reactions in an OpenArray 384-well Sample Plate 3 Set up the real-time PCR reactions in an OpenArray 384-well Sample Plate IMPORTANT! The 4 12 areas of the 384-well plate being filled must match the area designated in the QuantStudio 12K Flex Software for that set of samples. 1. Remove the OpenArray plate from the freezer; allow it to come to room temperature in its unopened sleeve (~15 minutes). The OpenArray plate must be completely thawed before transferring reactions to it from the 384-well sample plate. 2. Prepare a 1:20 dilution of the cdna template (the mir-amp reaction product) in 0.1X TE buffer. 3. Gently swirl the contents of the TaqMan OpenArray Real-Time PCR Master Mix to thoroughly mix. Do not invert the bottle. 4. Combine master mix and cdna samples in tubes, strip tubes, or a 96-well plate. Component Volume per well Volume per sample (16 subarrays) [1] TaqMan OpenArray Real- Time PCR Master Mix 2.5 µl 50 µl Diluted cdna template 2.5 µl 50 µl Total reaction volume 5.0 µl 100 µl [1] Volumes include 25% overage. 5. Following the plate layout designated in the EDS experiment file created in the QuantStudio 12K Flex Software, add 5.0 µl of the combined master mix and cdna sample to the sets of 4 4 wells in an OpenArray 384-well Sample Plate One full array 2 One 4 4 set of an array TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates) 17

18 3 Chapter 3 Prepare and run TaqMan Advanced mirna Assays with OpenArray plates Set up the real-time PCR reactions in an OpenArray 384-well Sample Plate If strip tubes or plates are used, load alternate wells to correspond to a 4 4 set of the array Samples added to alternating tubes or wells sets of an array 6. Thoroughly mix each PCR reaction by pipetting up and down or by using the "mix" function on the multi-channel pipette. 7. Seal the plate with an aluminum foil seal, remove the foil flap, mark the edges of the filled 4 12 area with a pen, then score the foil along those lines. Do not remove the foil from the scored area at this time. 8. Centrifuge the plate at 1,000 rpm for 1 minute. 18 TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates)

19 Chapter 3 Prepare and run TaqMan Advanced mirna Assays with OpenArray plates Set up the AccuFill instrument 3 Set up the AccuFill instrument IMPORTANT! Do not use OpenArray AccuFill System Tips that exceed the expiration date (shown on the outer box that contains the tip trays). 1. In the OpenArray AccuFill software, click Setup and Load. 2. In the Setup Load Information window, enter the OpenArray plate barcodes in the appropriate plate holder positions. Note: The Use Sample Integration checkbox should not be selected. 3. Click the corresponding 4 12 area of the 384-well plate, then click Next to open the Setup Deck window. TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates) 19

20 3 Chapter 3 Prepare and run TaqMan Advanced mirna Assays with OpenArray plates Transfer reactions to the OpenArray plate (AccuFill instrument) 4. Ensure that: Tip boxes are loaded in the AccuFill instrument in the displayed configuration. Lids are removed from the tip boxes. The waste bin in the instrument is emptied. 5. In the Setup Deck window, select: The tips are configured as shown above The Waste Bin is empty Transfer reactions to the OpenArray plate (AccuFill instrument) Ensure that the OpenArray plate is thawed and that the whole plate is at room temperature. 1. Prepare the items needed to seal the OpenArray plate (next section). Note: The OpenArray plate must be sealed promptly after being loaded with the reactions (this section). a. Ensure that the QuantStudio 12K Flex OpenArray Plate Press 2.0 is ready. b. Gather and remove from packaging an OpenArray lid, plug, syringe with OpenArray Immersion Fluid, and syringe tip. c. Attach the syringe tip to the syringe and carefully push some of the fluid through the tip to remove air bubbles, then lay the syringe aside. 2. Remove the OpenArray plate from its sleeve and place it in the plate holder of the AccuFill instrument. Ensure that the bar code on the OpenArray plate is facing left and the serial number is facing right. 3. Using forceps, peel the foil from the filled area of the OpenArray 384-well Sample Plate. 4. Close the instrument door. 20 TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates)

21 Chapter 3 Prepare and run TaqMan Advanced mirna Assays with OpenArray plates Seal the OpenArray plate 3 5. In the AccuFill software Setup Deck window, select the following confirmations, then click Load. The OpenArray Plate is in the Plate Holder Remove foil from the highlighted section of the Sample Plate 6. As soon as the Remove OpenArray Plate window appears, open the instrument door, then remove the loaded OpenArray plate. 7. Proceed immediately to seal the OpenArray plate (next section). Note: For best results, seal the OpenArray plate within 90 seconds of completion of loading, to prevent evaporation. Seal the OpenArray plate IMPORTANT! Handle the OpenArray plate and case only by the edges throughout this procedure. 1. Place the filled OpenArray plate in the QuantStudio 12K Flex OpenArray Plate Press 2.0. Ensure that the bar code is facing left and the serial number is facing right. 2. Remove the clear plastic sheets from the top and the bottom of the lid, remove the red protective film around the edge of the OpenArray lid, then seat the lid on the OpenArray case in the plate press Engage the press mechanism until the green flashing light changes to a steady green light (~20 seconds). 4. Disengage the press, then remove the OpenArray case. 1 Protective film (remove) 2 Adhesive 3 Protective film (remove) 4 Notched end (align with serial number) TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates) 21

22 3 Chapter 3 Prepare and run TaqMan Advanced mirna Assays with OpenArray plates Run the OpenArray plate(s) on the QuantStudio 12K Flex instrument 5. While holding the OpenArray case by the edges, insert the prepared syringe tip into the port in the case, then carefully inject immersion fluid until the case is filled. Note: Minimize creation of air bubbles when you dispense the fluid; one small air bubble in the case is acceptable. The syringe tip must be in front of the array when filling the case with immersion fluid. 6. While holding the case vertically, remove the syringe tip, insert the screw end of the OpenArray plug into the port and rotate clockwise until the black handle breaks off. IMPORTANT! To avoid leaking of immersion fluid, hold the case vertically and rotate the plug slowly. 7. Clean the case with a laboratory wipe that has been thoroughly sprayed with ethanol, then dry the case with a clean laboratory wipe. Run the OpenArray plate(s) on the QuantStudio 12K Flex instrument 1. On the instrument touchscreen, touch to extend the loading arm, and place the OpenArray plates on the plate adapter. Ensure that the plate barcode and serial number are facing the front of the instrument. 2. Touch to retract the loading arm. 3. In the Home screen of the QuantStudio 12K Flex Software, select Run4OpenArray. 4. In the Select Instrument pane, select your QuantStudio instrument. 5. Click Get Plate IDs to import the barcodes of the OpenArray plates. Once the OpenArray serial numbers appear, the loaded TPF files corresponding to each plate should appear in the Setup File field. If not, click Browse, then select the correct loaded TPF file from the Loaded EDT folder. 22 TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates)

23 Chapter 3 Prepare and run TaqMan Advanced mirna Assays with OpenArray plates Check the QC images 3 6. (Optional) Click Browse to change the QuantStudio Experiment File Location. 7. (Optional) Change the software-determined Experiment File Name. 8. Click Start Run. Note: The instrument pauses at 41 or 42 seconds prior to the end of the run. Wait for the system to complete the run before opening the EDS file. 9. Transfer the EDS file from the instrument to an accessible location for analysis. 10. Check the QC images for loading issues or leaks. Check the QC images Check the QC images before analysis. For additional information, see Appendix A, Troubleshooting. 1. In the QuantStudio 12K Flex Software Export screen: a. Click Browse to create a uniquely-named folder for the QC images export. b. Click Export QC Images (bottom of screen). IMPORTANT! Create a new folder for images each time; exporting a second run to the same folder overwrites the images. 2. View the following ROX image to check for loading quality issues: POST-READ_CHANNEL_4.tiff 3. Check for leaks or other displaced sample issues. a. View the following spotfinding images: s02_c001_t03_p0001_m1_x2_e1_cp#_spotfind.tiff s02_c040_t03_p0001_m1_x2_e1_cp#_spotfind.tiff Note: The cp# in the image file name refers to the array position (1 4) within the instrument. b. If a problem is found, view the following pre-run spotfinding image to determine if the issue existed even before cycling (this is useful for troubleshooting): s00_c001_t01_p0001_m2_x3_e1_cp#_spotfind.tiff 4. View the following FAM images to check for any fluorescent abnormalities and to confirm any problem seen in the spotfinding images: STAGE2_CYCLE1_CHANNEL_1.tiff STAGE2_CYCLE40_CHANNEL_1.tiff 5. Note any abnormalities found, as well as all other potentially relevant information related to the setup of the run. TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates) 23

24 4 Export and review TaqMan Advanced mirna Assay data Export data Prepare exported data for analysis Review results Export data 1. Open an EDS file in the QuantStudio 12K Flex Software. 2. In the Experiment Menu pane, click Export. 3. Click Load Export Set (bottom of the screen), select GE_export_setting, then click OK. 4. Select.xlsx from the File Type dropdown list (top-right of the screen). 5. (Optional) Perform any of the following actions to customize the file export: Select Open file(s) when export is complete. Click Browse to select a new Export File Location. Enter a new file name in the Export File Name text field. Click the Results tab, then select the content to export. 6. Click Start Export (bottom of the screen). If Open file(s) when export is complete is selected, then the file automatically opens. If the option is not selected, navigate to and open the exported XLSX file. Prepare exported data for analysis 1. Open the exported XLSX data file. 2. Ensure that the barcode, run conditions, and all selected data columns were exported correctly. 3. Scroll down to the data rows, select the headers and data, then copy-paste into a new worksheet. 4. Rename the new worksheet Data Table_Run File Name. 24 TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates)

25 Chapter 4 Export and review TaqMan Advanced mirna Assay data Review results 4 5. (Optional) To combine data from multiple OpenArray plates: a. Insert a Barcode column in the Data Table worksheet to track OpenArray barcodes. b. Copy-paste the barcode numbers to the appropriate cells in the new Barcode column. 6. Find-replace all "Undetermined" values with an empty cell (no value) in the C rt column. This step ensures an exact count of C rt values. 7. Delete rows that do not contain run data. Review results Note: These guidelines apply to results from experiments that included three or more technical replicates. Note: We encourage testing and establishing your own C rt cut-off value for each assay to achieve high sensitivity and specificity. 1. Review the exported data for through-hole results that may require special attention. 2. Consider filtering out from analysis through-holes with the following values: Parameter to examine Consider filtering out through-holes if C rt C rt >28 Amp Score Amp Score <1.24 Note: Through-holes with unexpected C rt values can also be identified by reviewing the Amplification Plot. 3. Review through-holes with C rt >28 and ensure that the C rt values are reproducible in all technical replicates. Note: C rt =28 is approximately equal to 1 copy of the target sequence in a reaction. 4. Take note of technical replicates with mean C rt <28 and a high standard deviation (>0.5). The data from these through-holes may require further review. 5. Ensure that at least half of the replicates amplified adequately and pass your review specifications. 6. Use your preferred method to analyze the data. TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates) 25

26 4 Chapter 4 Export and review TaqMan Advanced mirna Assay data Review results Fields for Pivot Tables To review results using pivot tables, you can use the following settings. Note: For the "Average of" and "StdDev of" summarizations, use the appropriate source field (C rt or Amp Score), then choose the calculation type. Area of pivot table Target-oriented view Fields to add Sample-oriented view Report Filter Sample Name [1] Column Labels Sample Name Row Labels Target Name Target Name Values Average of C rt Average of C rt StdDev of C rt [2] Count of C rt StdDev of C rt Count of C rt Average of Amp Score [1] To see individual sample results, select the sample from the dropdown list next to the Sample Name header. [2] A Values field will automatically appear in the Column Labels area. 26 TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates)

27 A Troubleshooting Troubleshoot with cycling and imaging run images (QC images) Many problems with OpenArray results can be diagnosed by examining the quality control (QC) images taken at various points during a cycling/imaging run. The QC images are fluorescent or reflected light images taken before, during, and after cycling. They may require adjustment to make image features visible. To view the images, we recommend that you install the free software program ImageJ, which allows you to easily manipulate the images in ways that other image viewers cannot. 1. In the QuantStudio 12K Flex Software Export screen: a. Click Browse to select a uniquely-named folder for the QC images export. b. Click Export QC Images (bottom of screen). IMPORTANT! Select a new folder for images each time; exporting a second run to the same folder overwrites the images. 2. Use ImageJ to view the images of interest. To... View image... Image description Confirm the identity of images within a folder Evaluate the loading quality Check for existing contamination on the case and/or heated cover Identify potential leaks or other contamination Look at patterns in the fluorescent data (for example, gradients) BARCODE IMAGE.tiff PRE-READ_CHANNEL_4.tiff POST-READ_CHANNEL_4.tiff s00_c001_t01_p0001_m2_x3_e1_cp#_spotfind.tiff [1] s02_c001_t03_p0001_m1_x2_e1_cp#_spotfind.tiff [1] s02_c040_t03_p0001_m1_x2_e1_cp#_spotfind.tiff [1] STAGEx_CYCLEy_CHANNEL_1.tiff Reflected light image of the entire OpenArray plate Pre- and post-rox images Pre-run reflected light spotfinding image (used by the software to determine the location of the holes) Mid-run reflected light spotfinding image Post-run reflected light spotfinding image FAM images at a particular cycle (y) of a particular stage (x) of the run. [1] The cp# in the image file name refers to the array position (1 4) within the QuantStudio 12K Flex instrument. 3. (Optional) Adjust the images for brightness and/or contrast to make image features visible. a. Open the image in ImageJ. TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates) 27

28 A Appendix A Troubleshooting AccuFill instrument plate loading errors b. Select Image4Adjust Brightness/Contrast (or press Ctrl+Shift+C). c. Click Auto or adjust the sliders until the features of interest in the image are visible. AccuFill instrument plate loading errors There are empty through-holes Turn-holes are repeatedly missed Observation Possible cause Recommended action Insufficient sample was added to the 384-well sample plate. Reaction mix (sample + master mix) is not at the bottom of the 384-well sample plate. AccuFill instrument is aligned too far to the left or to the right. Systematic loading problems can occur with the AccuFill instrument, which indicates a need for service. For example, when turn-holes are repeatedly missed across multiple subarrays, service is required. Turn holes are where the AccuFill instrument changes direction during sample loading. Use proper pipetting techniques. Ensure that there are no air bubbles in the pipette tips after sample aspiration. Centrifuge the sample plate at 1,000 rpm for 60 seconds. Contact your local field service engineer. Turn holes Start points Stop points 28 TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates)

29 Appendix A Troubleshooting OpenArray plate assembly and handling errors A Entire subarrays are missing Observation Possible cause Recommended action The sample/master mix was not added to particular wells in the 384-well sample plate. Stuck tip mandrel on AccuFill instrument may need cleaning. Pipette tip was not loaded on mandrel. Visually inspect the sample plate to ensure that the wells have sample/master mix. Contact your local field service engineer. Contact your local field service engineer for frequent occurrences (infrequent occurrences can be due to a poorly molded tip). OpenArray plate assembly and handling errors Observation Possible cause Recommended action Case leaks and bubbles inside the case Improper sealing of theopenarray plate in the OpenArray Case can lead to immersion fluid leaks or bubble formation inside the case, leading to uneven heating and imaging throughout PCR and to poor quality data. The images above are examples of OpenArray plates that have been affected by immersion fluid leaks. The images show where leaked fluid has condensed on the underside of the heated cover windows and obscured the view of the throughholes. Plate press was not engaged for at least 20 seconds. Damaged lid adhesive. Damaged fill port screw gasket. Damaged fill port screw assembly. Breaks off too easily. Oily lid or case from immersion fluid overflow. Immersion fluid was exposed to air for too long. Fully engage the plate press for at least 20 seconds. Remove the liner and visually inspect the lid adhesives for defects. Ensure that adhesive is not damaged or warped. Visually inspect the screw to ensure that the orange gasket is present and not damaged. The screw may be misthreaded: unscrew it and use a new screw assembly. Wipe off excess overflow of immersion fluid from the lid, case bottom, and crevices with 70% isopropyl alcohol, using a lint-free cloth (the cloth included with the OpenArray plate is acceptable). Do not remove the immersion fluid syringe cap or draw air bubbles into the syringe until you are ready to load. TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates) 29

30 A Appendix A Troubleshooting OpenArray plate assembly and handling errors Observation Possible cause Recommended action The best image in which to detect leaks is the s02_c001_t03_p0001_m1_x2_e1_cp#_spotfind.tiff image. This image is taken at the start of cycling, which is where most leaks occur. See Troubleshoot with cycling and imaging run images (QC images) on page 27. Sample blow-out during the addition of immersion fluid Evaporation of reaction mixture in through-holes Too large of a bubble inside the OpenArray case after sealing. Damaged plate press, leading to uneven pressure. The reactions in A12 were compromised during the addition of immersion fluid. Injecting the immersion fluid too quickly can purge the sample out of the throughholes near the fill port. Often this is caused by the user not purging the syringe slightly before use. Too much time elapsed before the plate was sealed with lid and immersion fluid. In this example, the top half of each subarray was intentionally left open to the environment to demonstrate the effect of evaporation. Donuts are a result of the evaporated fluid in the though-holes. Minimize the size of the bubble by tilting the OpenArray case so that the fill port is at the highest point. Slowly fill the case with immersion fluid until only a small air bubble remains. Attach the screw and wipe off any excess oil that may have spilled onto the case. Contact your field service engineer if you suspect that your plate press may be damaged. Dispense a small amount of immersion fluid onto a paper towel before use to ensure smooth operation of the syringe. Add immersion fluid as soon as the case is removed from the plate press to minimize the likelihood of evaporation, then seal the case with the lid. 30 TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates)

31 B Supplemental information Endogenous and exogenous controls cdna template preparation TaqMan Advanced mirna Assays chemistry overview Best practices for PCR and RT-PCR experiments Endogenous and exogenous controls Endogenous controls An endogenous control shows gene expression that is relatively constant and moderately abundant across treatment protocols, and tissues or cell types. Normalization to endogenous control genes is currently the most accurate method to correct for potential biases that are caused by: Sample collection Variation in the amount of starting material Reverse transcription (RT) efficiency Nucleic acid (RNA/DNA) preparation and quality No single control can act as a universal endogenous control for all experimental conditions, so we recommend verifying the chosen endogenous control or set of controls for the sample tissue, cell, or treatment. See Endogenous and exogenous controls on page 9 for available TaqMan Advanced mirna Assays that target mirnas with relatively constant expression levels across many different sample types. Exogenous controls An exogenous control is a synthetic RNA oligonucleotide with an mirna target sequence that is not present in the sample of interest. For example, the target sequence for the mirna assay ath-mir-159a is not present in humans, so it is a good exogenous control for human samples. The RNA oligonucleotide is combined with the biological sample during the RNA isolation procedure as a spike-in control to monitor: Sample input amount for difficult samples (for example, serum/plasma or other biofluids). Extraction efficiency. When using exogenous controls with TaqMan Advanced mirna Assays: The assay chemistry requires that exogenous controls be 5'-phosphorylated. The final concentration of the spike-in control in the sample should be 1 10 pm. See Table 6 on page 9 for available TaqMan Advanced mirna Assays which target sequences that can be used as exogenous controls with human samples. TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates) 31

32 B Appendix B Supplemental information cdna template preparation cdna template preparation Quantification using TaqMan Advanced mirna Assays requires the modification of mature mirnas by the addition of a poly(a) tail (3 ) and an adaptor (5 ) to: Amplify all mirnas in a single reverse transcription (RT) reaction. Amplify the sample for downstream PCR in a single universal cdna reaction. PAP 5' AAAAAAAAAAAA 3' mirna Poly(A) tail Poly(A) tailing reaction Starting with a total RNA sample, poly(a) polymerase is used to add a 3 adenosine tail to the mirna. Adaptor ligation reaction 5' Adaptor Lig AAAAAAAAAAAA 3' The mirna with poly(a) tail undergoes adaptor ligation at the 5 end. The adaptor acts as the forward-primer binding site for the mir-amp reaction. 5' AAAAAAAAAAAA 3' Reverse transcription (RT) reaction RT (V)TTTTTTTTTTTT A Universal RT primer binds to the 3 poly(a) tail and the mirna is reverse transcribed. The Universal RT primer resulting cdna is suitable for all TaqMan Advanced mirna Assays. mir-amp forward primer 3' P TTTTTTTTTTTT 5' 5' 3' P mir-amp reverse primer mir-amp reaction Universal forward and reverse primers increase the number of cdna molecules. LEGEND: PAP Poly(A) polymerase Lig Ligase RT Reverse Transcriptase P Hot-start DNA polymerase 32 TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates)

33 Appendix B Supplemental information TaqMan Advanced mirna Assays chemistry overview B TaqMan Advanced mirna Assays chemistry overview TaqMan MGB probes About the 5' nuclease assay TaqMan MGB probes contain: A reporter dye (for example, FAM ) at the 5 end of the probe (Afonina et al., 1997; Kutyavin et al., 1997). A non-fluorescent quencher (NFQ) dye at the 3 end of the probe. The NFQ dye does not fluoresce, which allows the real-time PCR system to measure the reporter dye contributions more accurately. A minor groove binder (MGB) at the 3 end of the probe that: Increases the melting temperature (T m ) without increasing the probe length. Allows for the design of shorter probes. Note: The following figures are general representations of real-time PCR with TaqMan MGB probes and TaqMan Advanced mirna Assays. The sequence regions are not necessarily drawn to scale. The 5' nuclease assay process takes place during PCR amplification. It occurs in every cycle and does not interfere with the exponential accumulation of product. Adaptor region mirna region Poly(A) tail region 3' 5' 5' 3' Figure 1 cdna synthesis product During the PCR, the forward and reverse primers anneal to complementary sequences along the denatured cdna template strands (Figure 2). The primer binding sites vary depending on the target mirna sequence and are designed to maximize specificity. Figure 2 shows an example representation in which the reverse primer is the primer that partially overlaps the mirna region. The TaqMan MGB probe anneals specifically to a complementary sequence between the forward and reverse primer sites (Figure 2). When the probe is intact, the proximity of the reporter dye and quencher dye suppresses the reporter fluorescence, primarily by Förster-type energy transfer. Forward primer R Probe NFQ MGB 3' 5' 5' 3' Reverse primer Figure 2 Annealing of probes and primers to cdna strands TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates) 33

34 B Appendix B Supplemental information TaqMan Advanced mirna Assays chemistry overview During polymerization, the DNA polymerase cleaves only probes that hybridize to the target sequence. Cleavage separates the reporter dye from the probe. The separation of the reporter dye from the quencher dye results in increased fluorescence by the reporter dye (Figure 3). This increase in fluorescence occurs only if the probe is complementary to the target sequence and if the target sequence is amplified during PCR. Because of these conditions, nonspecific amplification is not detected. R MGB NFQ P 3' 5' 5' 3' P Figure 3 Initial polymerization and cleavage of reporter dye Polymerization of the strand continues (Figure 4), but because the 3' end of the probe is blocked, no extension of the probe occurs during PCR. R NFQ MGB 3' 5' 5' 3' Figure 4 Completion of polymerization LEGEND: P Hot-start DNA polymerase R Reporter dye NFQ Non-fluorescent quencher dye MGB Minor groove binder 34 TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates)

35 Appendix B Supplemental information Best practices for PCR and RT-PCR experiments B Best practices for PCR and RT-PCR experiments Good laboratory practices for PCR and RT-PCR Use UNG to prevent falsepositive amplification When preparing samples for PCR or RT-PCR amplification: Wear clean gloves and a clean lab coat. Do not wear the same gloves and lab coat that you have previously used when handling amplified products or preparing samples. Change gloves if you suspect that they are contaminated. Maintain separate areas and dedicated equipment and supplies for: Sample preparation and reaction setup. Amplification and analysis of products. Do not bring amplified products into the reaction setup area. Open and close all sample tubes carefully. Avoid splashing or spraying samples. Keep reactions and components capped as much as possible. Use a positive-displacement pipettor or aerosol-resistant barrier pipette tips. Clean lab benches and equipment periodically with 10% bleach solution or DNAZap Solutions (Cat. No. AM9890). Carryover amplicons can result in false-positive amplification during PCR. Use a master mix that contains heat-labile uracil-n-glycosylase (UNG; also known as uracil-dna glycosylase (UDG)) to degrade many contaminating carryover amplicons. UNG enzymatic activity occurs during the PCR reaction setup at room temperature; an activation step before thermal cycling is not necessary. Unlike standard UNG, heat-labile UNG is completely inactivated during the first ramp to the hightemperature step for template denaturation and polymerase activation. To ensure the desired UNG activity: Use PCR components and thermal cycling conditions as specified. UNG-containing master mixes incorporate the optimal concentration of UNG to prevent cross-contamination while not affecting real-time PCR performance. Do not attempt to use UNG-containing master mixes in subsequent amplification of du-containing PCR products, such as in nested-pcr protocols. The UNG will degrade the du-containing PCR products, preventing further amplification. Although treatment with UNG can degrade or eliminate large numbers of carryover PCR products, use good laboratory practices to minimize cross-contamination from non-du-containing PCR products or other samples. Detect fluorescent contaminants Fluorescent contaminants can generate false positive results. To help detect these contaminants, we recommend including a No-Amplification Control reaction that contains sample, but no master mix. After PCR, if the absolute fluorescence of the No-Amplification Control is greater than the fluorescence of the no template control (NTC), fluorescent contaminants may be present in the sample or in the heat block of the real-time PCR instrument. TaqMan Advanced mirna Assays User Guide (TaqMan OpenArray Plates) 35