PROTOCOL. TaqMan OpenArray microrna Panel Using Megaplex Primer Pools

Transcription

1 PROTOCOL TaqMan OpenArray microrna Panel Using Megaplex Primer Pools Publication Part Number Rev. B Revision Date March 2011

2 For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. Information in this document is subject to change without notice. APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. TO THE FULLEST EXTENT ALLOWED BY LAW, IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF, WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES. NOTICE TO PURCHASER: Limited Use Label License The product(s) shown in this presentation may be covered by one or more Limited Use Label License(s). Please refer to the respective product documentation or the Applied Biosystems website under for the comprehensive license information. By use of these products, the purchaser accepts the terms and conditions of all applicable Limited Use Label Licenses. These products are sold for research use only, and are not intended for human or animal diagnostic or therapeutic uses unless otherwise specifically indicated in the applicable product documentation or the respective Limited Use Label License(s). For information on obtaining additional rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California Research Use Only The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial applications of any kind, including, without limitation, quality control and commercial services such as reporting the results of purchaser's activities for a fee or other form of consideration. For information on obtaining additional rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. TaqMan is a registered trademark of Roche Molecular Systems, Inc. Integromics and RealTime StatMiner are registered trademarks of Integromics, S.L Life Technologies Corporation Corporation. All rights reserved.

3 Contents About This Guide Purpose Safety information Safety alert words SDSs PROTOCOL TaqMan OpenArray Human microrna Panel Product information Purpose of the product Contents and storage Materials and equipment required but not included Workflow Plate overview MicroAmp Optical 96-Well Reaction Plate OpenArray 384-Well Reaction Plate TaqMan OpenArray microrna Panel Plate loading scheme Run the Megaplex Pools with PreAmplification Prepare the sample Run the Megaplex reactions Run the PreAmplification reactions Run the OpenArray Panels Analyze the data Troubleshoot APPENDIX A Ordering Information How to order Materials and equipment not included Megaplex Primer Pools Reagents Laboratory equipment APPENDIX B PCR Good Laboratory Practices TaqMan OpenArray mirna Panel Protocol 3

4 Contents APPENDIX C Safety Chemical safety General chemical safety SDSs Biological hazard safety Documentation and Support Related documentation OpenArray Real-Time PCR System Obtaining support TaqMan OpenArray mirna Panel Protocol

5 About This Guide Purpose The TaqMan OpenArray Human microrna Panel Protocol provides instructions and troubleshooting information for using the TaqMan OpenArray microrna Panels and Megaplex Primer Pools. Safety information Note: For general safety information, see this section and Appendix C, Safety on page 27. When a hazard symbol and hazard type appear by a chemical name or instrument hazard, see the Safety Appendix for the complete alert on the chemical or instrument. Safety alert words Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards. Each alert word IMPORTANT, CAUTION, WARNING, DANGER implies a particular level of observation or action, as defined below: IMPORTANT! Indicates information that is necessary for proper instrument operation, accurate chemistry kit use, or safe use of a chemical. CAUTION! Indicates a potentially hazardous situation that, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices. WARNING! Indicates a potentially hazardous situation that, if not avoided, could result in death or serious injury. DANGER! Indicates an imminently hazardous situation that, if not avoided, will result in death or serious injury. This signal word is to be limited to the most extreme situations. TaqMan OpenArray Human microrna Panel Protocol 5

6 About This Guide Safety information SDSs The Safety Data Sheets (SDSs) for any chemicals supplied by Applied Biosystems or Ambion are available to you free 24 hours a day. For instructions on obtaining SDSs, see SDSs on page 27. IMPORTANT! For the SDSs of chemicals not distributed by Applied Biosystems or Ambion contact the chemical manufacturer. 6 TaqMan OpenArray Human microrna Panel Protocol

7 PROTOCOL TaqMan OpenArray Human microrna Panel Product information Purpose of the product Megaplex Primer Pools are designed to detect and quantitate up to 377 micrornas per pool (pools A and B) in either human, mouse, or rat species using Applied Biosystems real-time instruments. Megaplex Primer Pools consist of matching pools and TaqMan OpenArray microrna panels: Megaplex RT Primers is a set of two predefined pools (Pool A and Pool B) of up to 381 stem-looped reverse-transcription (RT) primers per pool that enable the simultaneous synthesis of cdna for mature micrornas. Megaplex PreAmp Primers is a set of two pools (Pool A and Pool B) of gene-specific forward and reverse primers. The primers enable the unbiased preamplification of the microrna cdna target by PCR prior to loading the TaqMan OpenArray Plate. TaqMan OpenArray Plate for microrna is a 3072-well microfluidic plate containing dried TaqMan primers and probes. The array enables quantitation of the gene expression levels of up to 758 micrornas and controls. This is accomplished by loading the preamplified cdna product onto the array for PCR amplification and realtime analysis. Each TaqMan OpenArray Plate is matched to a downloadable OpenArray Plate File (in.tpf format), available online at: Contents and storage TaqMan OpenArray Part Number Contents TaqMan OpenArray Human microrna Panel A single OpenArray Plate sufficient for three samples The TaqMan OpenArray Plates are shipped on dry ice. When you receive the arrays, store them at 15 to 25 C and keep them protected from light. The Megaplex Primer Pools are shipped at ambient temperature. When you receive the pools, store them at 15 to 25 C. TaqMan OpenArray mirna Panel Protocol 7

8 TaqMan OpenArray Human microrna Panel Materials and equipment required but not included Materials and equipment required but not included For optional materials and equipment and more ordering information, see Appendix A, Ordering Information on page 23. Megaplex RT and PreAmp Primers Part Number Contents Human Set This part number corresponds to four kits: Megaplex RT Pool A Megaplex RT Pool B Megaplex PreAmp Primers, Pool A 50 µl of stem-loop RT primers, 1000 µl 25 mm MgCl µl of forward and reverse primers Megaplex PreAmp Primers, Pool B Item Source TaqMan OpenArray Real-Time PCR Master Mix, 1.5 ml Part no TaqMan OpenArray Real-Time PCR Master Mix, 5 ml Part no mirvana microrna Isolation Kit Part no. AM1560 TaqMan MicroRNA Reverse Transcription Kit, 1000 rxns Part no TaqMan MicroRNA Reverse Transcription Kit, 200 rxns Part no TaqMan PreAmp Master Mix Part no OpenArray Real-Time PCR Accessories Kit Part no OpenArray 384-well Sample Plates Part no OpenArray 384-Well Barcoded Sample Plates Part no MicroAmp Clear Adhesive Film Part no MicroAmp Optical 96-Well Reaction Plate Part no Lab equipment MLS For the SDS of any chemical not distributed by Applied Biosystems, contact the chemical manufacturer. Before handling any chemicals, refer to the SDS provided by the manufacturer, and observe all relevant precautions. You will also need one of the following systems to load the OpenArray Plates: Item Source OpenArray AccuFill System Part no OpenArray AccuFill System Tips Part no OpenArray AccuFill System Tips, 10 Pack Part no Can be substituted by the OpenArray Autoloader (Part no ) and associated tips. 8 TaqMan OpenArray mirna Panel Protocol

9 TaqMan OpenArray Human microrna Panel Workflow Workflow Run the Megaplex Pools with PreAmplification (page 14) Prepare the sample (page 14) Prepare the RT Reaction Mix (page 14) Run the Megaplex RT reactions (page 15) (Optional) Stopping Point (page 15) Run the PreAmplification reactions (page 16) Prepare the PreAmp Reaction Mix (page 16) Perform the PreAmplification (page 17) Dilute the PreAmplification reaction (page 17) (Optional) Stopping Point (page 17) Run the OpenArray Panels (page 17) Access the OpenArray Plate files (page 18) Prepare the TaqMan OpenArray microrna Panel (page 18) Prepare the PCR Reaction Mix (page 18) Load and run the OpenArray Plate (page 18) Analyze the data (page 20) TaqMan OpenArray mirna Panel Protocol 9

10 TaqMan OpenArray Human microrna Panel Workflow For a full microrna profile, run two Megaplex RT reactions (Pool A and B) and two preamplification reactions (Pool A and B) per sample. A full microrna profile takes one-third of an OpenArray Plate, and a full OpenArray Plate can accommodate up to three individual samples. Alternatively, a full OpenArray Plate can be used to run a sample with three replicates. 10 TaqMan OpenArray mirna Panel Protocol

11 TaqMan OpenArray Human microrna Panel Plate overview Plate overview This protocol requires three types of plates: MicroAmp Optical 96-Well Reaction Plate (96-well plate) OpenArray 384-Well Sample Plate (384-well plate) TaqMan OpenArray microrna Panel (OpenArray Plate) (page 12) MicroAmp Optical 96-Well Reaction Plate This is a 96-well thermocycling reaction plate. In this protocol, 96-well plates are used in the following procedures: Run the Megaplex RT reactions (page 15) Run the PreAmplification reactions (two plates) (page 16) Dilute the PreAmplification reaction (page 17) Run the OpenArray Panels (page 17) OpenArray 384-Well Reaction Plate In this protocol, 384-well plates are used to transfer the PreAmplification product and Master Mix into the OpenArray Plate using the OpenArray AccuFill Platform (page 17). The 384-well plate can be divided into eight regions, each containing 48 wells. Each 48- well region is used to fill one OpenArray Plate. Each sample well on the 384-well plate is used to load a subarray of 64 through-holes on the OpenArray Plate (see the illustration of the plate on page 12). TaqMan OpenArray mirna Panel Protocol 11

12 TaqMan OpenArray Human microrna Panel Plate overview IMPORTANT! The well dimensions of the OpenArray 384-Well Sample Plates are specifically suited for use with the AccuFill system. We do not recommend the use of other microtiter plates. TaqMan OpenArray microrna Panel The TaqMan OpenArray microrna Panel is a 63 mm 19 mm mid-density reaction plate. There are 3072 through-holes in the plate, of which 2688 are preloaded with TaqMan primers and probes. Individual through-holes accommodate 33-nL reaction volumes. As shown in the following figure, the OpenArray Plate is divided into 48 subarrays; each subarray consists of 64 through-holes. Hydrophilic and hydrophobic coatings enable reagents to be held within the through-holes. An entire sub-array (64 throughholes) is loaded from each sample well in the 384-well sample plate using the AccuFill system. Plate loading scheme The plates are loaded according to the capacity of a single OpenArray Plate. Each OpenArray Plate can accommodate three samples (or one sample in triplicate) for a full microrna profile against Megaplex Primer Pools A and B. We recommend using 96-well plates to perform sample preparation and mixing for the RT and preamplification reactions, and OpenArray 384-well plates to prepare the PCR reaction mix for loading into the OpenArray Plate. A single 96-well sample plate can hold up to 48 samples (each run against Pool A and Pool B). These 48 samples would be sufficient to fill two 384-well plates, which in turn will load a total of 16 OpenArray Plates. Thus, the plate loading scheme is illustrated on page TaqMan OpenArray mirna Panel Protocol

13 TaqMan OpenArray Human microrna Panel Plate overview OpenArray keep ths for xref to fig TaqMan OpenArray mirna Panel Protocol 13

14 TaqMan OpenArray Human microrna Panel Run the Megaplex Pools with PreAmplification Run the Megaplex Pools with PreAmplification Prepare the sample Run the Megaplex reactions The recommended starting material is total RNA or cell lysate. To prepare the starting material, use the recommended kit for your application. For each Megaplex RT pool, the RT reaction with downstream preamplification supports ng of input total RNA. For most tissues, 100 ng of total RNA produces a comprehensive microrna profile. We recommend verifying the optimal quantity of input total RNA for your sample type. IMPORTANT! The method used to isolate the total RNA must preserve the small RNA fraction. However, in order to preserve endogenous control sequences in the total RNA, do not enrich for the small RNA fraction. This may cause longer control transcripts (snornas) to be lost. For a full microrna profile, run two Megaplex RT reactions (Pool A and B) and two Megaplex preamplification reactions (Pool A and B) per sample. You can run up to three full microrna profiles (that is, a full profile on three samples or one sample in triplicate) in a single OpenArray Plate. Prepare the RT Reaction Mix In this step, use the TaqMan MicroRNA Reverse Transcription Kit and the Megaplex RT Primers to synthesize single-stranded cdna from total RNA or cell lysate samples. (See Appendix A on page 23 for part numbers.) 1. Thaw the following on ice: Megaplex RT Primers TaqMan MicroRNA Reverse Transcription Kit components MgCl 2 (supplied with the Megaplex RT Primers) 2. Combine the following in each of two 1.5-mL microcentrifuge tubes (one for Pool A, the other for Pool B): RT Reaction Mix Components Volume for One Sample Volume for Nine Samples Megaplex RT Primers (10 ) 0.75 µl 7.6 µl dntps with dttp (100 mm) 0.15 µl 1.5 µl MultiScribe Reverse Transcriptase (50 U/µL) 1.50 µl 15.2 µl 10 RT Buffer 0.75 µl 7.6 µl MgCl 2 (25 mm) 0.90 µl 9.1 µl RNase Inhibitor (20 U/µL) 0.09 µl 0.91 µl Nuclease-free water 0.35 µl 3.54 µl Total 4.50 µl Use Pool A in one tube, and Pool B in the other. Includes 12.5% excess for loss from pipetting. This is sufficient for three OpenArray Plates with three samples each. 14 TaqMan OpenArray mirna Panel Protocol

15 TaqMan OpenArray Human microrna Panel Run the Megaplex Pools with PreAmplification 3. Pipet six times to mix, then centrifuge the tubes briefly. 4. Pipet 4.5 µl of the RT reaction mix into each well of a 96-well MicroAmp Optical Reaction Plate. Note: Remember that each sample is processed in two wells: one for Pool A and one for Pool B. Thus each 96-well plate can process 48 samples. See Plate loading scheme on page Add 3 µl of solution (containing 100 ng of total RNA, recommended) into each well containing RT reaction mix. Note: You can use 3 µl of water for the No Template Control reactions. 6. Seal the plate using MicroAmp Clear Adhesive Film. Then invert the plate six times to mix. Spin briefly. Note: Do not use MicroAmp Optical Adhesive Film to seal the plate. 7. Incubate the plate on ice for 5 minutes. Run the Megaplex RT reactions About the RT reaction The reverse transcription (RT) reaction has a final volume of 7.5 µl and contains: 3 µl of solution (containing 100 ng of total RNA, recommended) 4.5 µl of RT reaction mix Perform Megaplex reverse transcription 1. Set up the run method using the following conditions: Ramp speed or mode: 9700 using Std or Max ramp speed. Reaction volume (µl): 7.5 Thermal-cycling conditions: Stage Temp Time Cycle (40 Cycles) 16 C 2 min 42 C 1 min 50 C 1 sec Hold 85 C 5 min Hold 4 C 2. Load, then run the plate. STOPPING POINT The cdna can be stored at 15 to 25 C for at least one week. TaqMan OpenArray mirna Panel Protocol 15

16 TaqMan OpenArray Human microrna Panel Run the Megaplex Pools with PreAmplification Run the PreAmplification reactions About the PreAmplification reaction In this step, preamplify specific cdna targets to increase the quantity of desired cdna for gene expression analysis using TaqMan OpenArray microrna Panels. The preamplification reaction has a final volume of 25 µl and contains: 2.5 µl RT product 22.5 µl PreAmp reaction mix Prepare the PreAmp Reaction Mix 1. Thaw the Megaplex PreAmp Primers on ice and mix by inverting six times. Spin briefly. 2. Mix the TaqMan PreAmp Master Mix (2 ) by swirling the bottle. 3. Combine the following in each of two 1.5-mL microcentrifuge tubes (one for Pool A, the other for Pool B): PreAmp Reaction Mix Components Volume for One Sample Volume for Nine Samples TaqMan PreAmp Master Mix (2 ) 12.5 µl µl Megaplex PreAmp Primers (10 ) 2.5 µl 25.3 µl Nuclease-free water 7.5 µl 75.9 µl Total 22.5 µl µl Use Pool A in one tube, and Pool B in the other. Includes 12.5% excess for volume loss from pippetting. 4. Pipet the tubes six times to mix, then centrifuge the tubes briefly. 5. In a 96-well MicroAmp Optical Reaction Plate, pipet 2.5 µl of each RT product (from Perform Megaplex reverse transcription on page 15) into its corresponding wells. 6. Dispense 22.5 µl of PreAmp reaction mix into each well of the 96-well plate containing the RT product. 7. Seal the plate using MicroAmp Clear Adhesive Film. Invert the plate six times to mix. Spin briefly. 8. Incubate the plate on ice for 5 minutes. 16 TaqMan OpenArray mirna Panel Protocol

17 TaqMan OpenArray Human microrna Panel Run the Megaplex Pools with PreAmplification Perform the PreAmplification Set up the run method using the following conditions: Ramp speed or mode: 9700 using Std ramp speed. Reaction volume (µl): 25 Thermal-cycling parameters: Stage Temp Time Hold 95 C 10 min Hold 55 C 2 min Hold 72 C 2 min Cycle (12 Cycles) Hold 95 C 15 sec 60 C 4 min 99.9 C 10 min Hold 4 C Required for enzyme inactivation. Dilute the PreAmplification reaction 1. Remove the 96-well plate from the thermal cycler. 2. Briefly centrifuge the plate. 3. For each sample, add 156 µl of 0.1 TE ph 8.0 to one well of a 96-well plate. 4. Transfer 4 µl of the preamplified cdna solution to each well containing TE buffer (final dilution: 1 to 40). Note: The diluted preamplification product is stable for up to 12 hours at 4 C, and is sufficient for up to nine samples. 5. Seal the 96-well plate containing the diluted samples. Invert the plate six times to mix, then centrifuge the plate briefly. 6. Proceed directly to Run the OpenArray Panels on page 17. STOPPING POINT Store the preamplified product (diluted or undiluted) at 15 to 25 C for up to one week, or place the product on ice and proceed directly to Run the OpenArray Panels on page 17. Run the OpenArray Panels About the Real-Time PCR Reaction In this step, the DNA polymerase from the TaqMan OpenArray Real-Time PCR Master Mix amplifies the preamplified target cdna using sequence-specific primers and probe on the TaqMan OpenArray Plate. The presence of the target is measured in real time through cleavage of the TaqMan probe by the polymerase 5-3 exonuclease activity and detection of the resulting increase in fluorescence. TaqMan OpenArray mirna Panel Protocol 17

18 TaqMan OpenArray Human microrna Panel Run the Megaplex Pools with PreAmplification Access the OpenArray Plate files Prepare the TaqMan OpenArray microrna Panel Prepare the PCR Reaction Mix Locate the plate file for each OpenArray Plate by downloading from: The barcode from one OpenArray Plate per order is required to download the correct files. Remove the TaqMan OpenArray Plate from the freezer and allow it to come to room temperature. This should take approximately 15 minutes. Note: All volumes in this section are calculated to include 12.5% excess volume loss from pipetting. 1. Thaw the diluted, stored PreAmp product (from Dilute the PreAmplification reaction on page 17) completely, on ice. Mix by inverting six times, then centrifuge the plate briefly. Note: We recommend storing TaqMan OpenArray Real-Time PCR Master Mix at -15 C to -25 C until first use. If frozen, fully thaw on ice prior to use. 2. Mix the TaqMan OpenArray Real-Time PCR Master Mix by swirling the bottle. 3. Pipet 22.5 µl of PCR Master Mix into each of two adjacent wells per sample (one for Pool A and one for Pool B) of a clean 96-well plate. See the Plate loading scheme on page 12 for detailed layout recommendations. 4. For each sample, pipet 22.5 µl of diluted Pool A PreAmp into one well of each pair, and 22.5 µl of diluted Pool B PreAmp into the other well. Refer to Load and run the OpenArray Plate for detailed Note: We recommend that you use the PCR Reaction Mix within one hour. It is sufficient for up to three samples. 5. Seal the plate, vortex gently to mix, and centrifuge the plate briefly. Load and run the OpenArray Plate For detailed information loading and sealing the OpenArray Plate using AccuFill, refer to the OpenArray Real-Time PCR Plates Protocol (Part no ). For information on running PCR with the Open Array plate, refer to the OpenArray Real-Time PCR System User Guide (Part no ). 1. After the TaqMan OpenArray Plate has reached room temperature, carefully remove it from its packaging. 18 TaqMan OpenArray mirna Panel Protocol

19 TaqMan OpenArray Human microrna Panel Run the Megaplex Pools with PreAmplification 2. For each sample and primer pool set (pool A or pool B), aliquot 5 µl of each reaction mixture into each of eight wells on an OpenArray 384-well sample plate, as shown on the plate map below (each square represents eight sample wells). Use a second plate if needed to hold all samples. Refer to the Plate loading scheme on page Load the OpenArray Plates from the sample plate, using the standard AccuFill method. See the OpenArray AccuFill System User Guide (Part no ). 4. Run the OpenArray Plates using the downloaded.tpf file corresponding to the barcode for each plate. Note: OpenArray Plates must be run within one hour of loading. TaqMan OpenArray mirna Panel Protocol 19

20 TaqMan OpenArray Human microrna Panel Analyze the data Analyze the data For detailed information on how to analyze and export primary real-time data, refer to the OpenArray Real-Time PCR System User Guide (Part no ). Export the data from the OpenArray Real-Time qpcr Analysis Software following procedures outlined in the OpenArray Real-Time PCR System User Guide (Part no ). Export summary results: Select File > Export Cycling Data. Select the Summary Results checkbox in the dialog box. Perform the analysis with DataAssist Software or Integromics RealTime StatMiner software. DataAssist software is a free download from the Applied Biosystems website. Go to and select Products > Real-Time PCR > Software > DataAssist Software. 20 TaqMan OpenArray mirna Panel Protocol

21 TaqMan OpenArray Human microrna Panel Troubleshoot Troubleshoot Observation Possible Cause Recommended Action The C t value for the No Template Control (NTC) is < 25 for some assays The C t values for the endogenous controls are highly variable across the sample set No amplification or poor amplification of samples Poor reproducibility across technical replicates Non-specific interactions between primers. Contamination Improper dilution of the preamplification reaction prior to real-time PCR Natural biological variation Low-abundance gene Use of non-applied Biosystems reagents Omission of reaction components, or deviation from the recommended protocol Sample omitted from RT reaction Inadequate mixing of reagents For NTC information on a specific assay, refer to the Megaplex Assay Performance File. Work in an amplicon-free environment. Follow the recommendations in the protocol. See Dilute the PreAmplification reaction on page 17. Use an alternative endogenous control such as a non-variable microrna. Use an alternative normalization strategy. Use more starting material. Use Applied Biosystems reagents. Add all required reaction components. Follow precise recommendations in protocol. Add sample to RT reaction. Make sure that all samples and reactions are mixed well by inverting six times as described in the protocol. TaqMan OpenArray mirna Panel Protocol 21

22 TaqMan OpenArray Human microrna Panel Troubleshoot 22 TaqMan OpenArray mirna Panel Protocol

23 APPENDIX A Ordering Information How to order For information on the TaqMan Human OpenArray Plates, go to the Applied Biosystems website at and select: Products > Gene Expression > microrna Assays. TaqMan OpenArray Part Number Contents TaqMan Human OpenArray microrna Panel Single OpenArray Panel sufficient for 3 samples Materials and equipment not included Megaplex Primer Pools Megaplex RT and PreAmp Primers Part Number Contents Human Set Each part number corresponds to four kits: Megaplex RT Pool A Megaplex RT Pool B Megaplex PreAmp Primers, Pool A 50 µl of Megaplex RT primers, 1000 µl 25 mm MgCl µl of Megaplex PreAmp primers. Megaplex PreAmp Primers, Pool B Reagents Item TaqMan OpenArray Real-Time PCR Master Mix mirvana microrna Isolation Kit Source Applied Biosystems Part no. TBD Part no. AM1560 TaqMan MicroRNA Reverse Transcription Kit, 1000 rxns Part no TaqMan OpenArray mirna Panel Protocol 23

24 Appendix A Ordering Information Materials and equipment not included Item Source TaqMan MicroRNA Reverse Transcription Kit, 200 rxns Part no TaqMan PreAmp Master Mix Part no Laboratory equipment Item Source OpenArray Real-Time PCR Accessories Kit Part no OpenArray 384-well Sample Plates Part no Lab equipment MLS For the SDS of any chemical not distributed by Applied Biosystems, contact the chemical manufacturer. Before handling any chemicals, refer to the SDS provided by the manufacturer, and observe all relevant precautions. You will also need one of the following systems to load the OpenArray Plates: Item Source OpenArray AccuFill System OpenArray AccuFill System Tips OpenArray AccuFill System Tips, 10 Pack Can be substituted by the OpenArray Autoloader (Part no ) 24 TaqMan OpenArray mirna Panel Protocol

25 APPENDIX B PCR Good Laboratory Practices When preparing samples for PCR amplification: Use a positive-displacement pipette or aerosol-resistant pipette tips. Follow proper pipette-dispensing techniques to prevent aerosols. Wear clean gloves and a clean lab coat (not previously worn while handling amplified PCR products or used during sample preparation). Change gloves whenever you suspect that they are contaminated. Maintain separate areas and dedicated equipment and supplies for: Sample preparation PCR setup PCR amplification Analysis of PCR products Never bring amplified PCR products into the PCR setup area. Open and close all sample tubes carefully. Centrifuge tubes before opening. Try not to splash or spray PCR samples. Keep reactions and components capped as much as possible. Clean lab benches and equipment periodically with 10% bleach solution. Use DNAZap Solution (Part no. AM9890). TaqMan OpenArray mirna Panel Protocol 25

26 Appendix B PCR Good Laboratory Practices 26 TaqMan OpenArray mirna Panel Protocol

27 APPENDIX C Safety This appendix covers: Chemical safety General chemical safety SDSs Biological hazard safety Chemical safety General chemical safety Chemical hazard warning WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below, and consult the relevant SDS for specific precautions and instructions: Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. (See About SDSs for information on ordering SDSs.) Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, a fume hood). Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer's cleanup procedures as recommended in the SDS. Comply with all local, state/provincial, or national laws and regulations related to chemical storage, handling, and disposal. SDSs About SDSs Chemical manufacturers supply current Safety Data Sheets (SDSs) with shipments of hazardous chemicals to new customers. They also provide SDSs with the first shipment of a hazardous chemical to a customer after an SDS has been updated. SDSs provide the safety information you need to store, handle, transport, and dispose of the chemicals safely. Each time you receive a new SDS packaged with a hazardous chemical, be sure to replace the appropriate SDS in your files. TaqMan OpenArray mirna Panel Protocol 27

28 Appendix C Safety Chemical safety Obtaining SDSs The SDS for any chemical supplied by Applied Biosystems is available to you free 24 hours a day. To obtain SDSs: 1. Go to click Support, then select SDS. 2. In the Keyword Search field, enter the chemical name, product name, SDS part number, or other information that appears in the SDS of interest. Select the language of your choice, then click Search. 3. Find the document of interest, right-click the document title, then select any of the following: Open To view the document Print Target To print the document Save Target As To download a PDF version of the document to a destination that you choose Note: For the SDSs of chemicals not distributed by Applied Biosystems, contact the chemical manufacturer. Biological hazard safety General biohazard WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Follow all applicable local, state/provincial, and/or national regulations. Wear appropriate protective equipment, which includes but is not limited to: protective eyewear, face shield, clothing/lab coat, and gloves. All work should be conducted in properly equipped facilities using the appropriate safety equipment (for example, physical containment devices). Individuals should be trained according to applicable regulatory and company/ institution requirements before working with potentially infectious materials. Read and follow the applicable guidelines and/or regulatory requirements in the following: U.S. Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories (stock no ; Occupational Safety and Health Standards, Bloodborne Pathogens (29 CFR ; nara/cfr/waisidx_01/ 29cfr1910a_01.html). Your company s/institution s Biosafety Program protocols for working with/ handling potentially infectious materials. Additional information about biohazard guidelines is available at: 28 TaqMan OpenArray mirna Panel Protocol

29 Documentation and Support Related documentation The following related documents are shipped with the system, and provide additional information about the use of the TaqMan OpenArray MicroRNA Panel: Document Part Number Description TaqMan OpenArray Human microrna Panel Quick Reference Card OpenArray Plates Product Insert Provides brief, step-by-step procedures for preparing total RNA and cell lysate samples for PCR on OpenArray Plates. It is designed to help you quickly learn to use the OpenArray Human microrna Panel Provides inventory and storage information for OpenArray Plates. Note: For additional documentation, see Obtaining support on page 30. OpenArray Real-Time PCR System The following documents provide information about other components of the OpenArray Real-Time PCR System. These components are required for the use of the OpenArray plates. Document OpenArray Real-Time PCR System Troubleshooting Guide OpenArray Real-Time PCR System User Guide OpenArray Real-Time PCR System Quick Reference Card: Workflow OpenArray Real-Time PCR System Quick Reference Card: Using the Sample Tracking Tool TaqMan OpenArray Real-Time PCR Plates Protocol Description Provides troubleshooting information for the OpenArray Real-Time PCR System. Provides procedures for imaging and analyzing OpenArray Plates. Provides maintenance information for the OpenArray Real-Time PCR System. Describes the overall workflow and provides brief procedures for performing gene expression experiments with the OpenArray Real- Time PCR System. Describes how to use the Sample Tracking Tool to create sample information files (.csv) and import the files into the OpenArray Real- Time qpcr Analysis Software. Provides procedures for preparing the TaqMan OpenArray Real- Time PCR Plates. Part Number TaqMan OpenArray mirna Panel Protocol 29

30 Documentation and Support Obtaining support Document OpenArray AccuFill System User Guide Description Provides procedures for using the OpenArray AccuFill System to load TaqMan OpenArray Real-Time PCR Plates Part Number Obtaining support For the latest services and support information for all locations, go to: At the Life Technologies web site, you can: Access worldwide telephone and fax numbers to contact Life Technologies Technical Support and Sales facilities. Search through frequently asked questions (FAQs). Submit a question directly to Technical Support. Order Life Technologies user documents, SDSs, certificates of analysis, and other related documents. Download PDF documents. Obtain information about customer training. Download software updates and patches. 30 TaqMan OpenArray mirna Panel Protocol

31 Part Number Rev. B 3/2011 Applied Biosystems 850 Lincoln Centre Drive Foster City, CA USA Phone Toll Free Technical Resources and Support For the latest technical resources and support information for all locations, please refer to our Web site at