High Capacity RNA-to-cDNA TM Master Mix. Protocol

Transcription

1 High Capacity RNA-to-cDNA TM Master Mix Protocol

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. Limited Use Label License This product is covered by US patent 7,470,515, US patent 7,638,612 and other patents pending in the United States and Europe. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer. The buyer is not authorized to sell or otherwise transfer this product, any of its components to a third party. The purchase of this product does not authorize the purchaser to use the product or any of its components for manufacture of commercial product. For information on obtaining a license to this product for purposes other than research, contact Licensing Department, Quanta BioSciences, Inc., 202 Perry Parkway, Suite 1, Gaithersburg, MD 20877; Telephone number: 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. Windows and Excel are registered trademarks of Microsoft Corporation in the United States and other countries. Corning is a trademark of Corning Inc. Ghost is a trademark of Symantec Corporation Life Technologies Corporation. All rights reserved. Part Number Rev. C 06/2010

3 Contents Preface Safety Safety Alert Words Chemical Hazard Warning Chemical Safety Guidelines About SDSs Obtaining SDSs Chemical Waste Hazards Chemical Waste Safety Guidelines Waste Disposal Biological Hazard Safety How to Obtain Support Master Mix Supplies and Procedures Overview Purpose of the High Capacity RNA-to-cDNA Master Mix About This Protocol Good Laboratory Practices PCR Good Laboratory Practices Bibliography Materials and Equipment Available Kits High Capacity RNA-to-cDNA Master Mix Components Storage of Master Mix Equipment Required but Not Supplied Materials Required but Not Supplied Using the High Capacity RNA-to-cDNA Master Mix Overview Diagram Optimizing the RNA Template

4 Contents Amount of RNA Preparing the Reverse Transcription Reactions with High Capacity RNA-to-cDNA Master Mix Performing Reverse Transcription with High Capacity RNA-to-cDNA Master Mix Storing cdna Reverse Transcription Reactions Appendix A: Examples of cdna Yields from Reverse Transcription Quantitative PCR Yields for Different Targets Index

5 Preface This preface covers: Safety How to Obtain Support Safety Safety Alert Words Four safety alert words appear in 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. Definitions IMPORTANT! Indicates information that is necessary for proper instrument operation, accurate chemistry kit use, or safe use of a chemical. 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. Indicates a potentially hazardous situation that, if not avoided, could result in death or serious injury. 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. Chemical Hazard Warning CHEMICAL HAZARD. Some of the chemicals used with instruments and protocols are potentially hazardous and can cause injury, illness, or death. 5

6 Preface Chemical Safety Guidelines About SDSs Obtaining SDSs To minimize the hazards of chemicals: 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 on page 6.) Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). For additional safety guidelines, consult the SDS. Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood). For additional safety guidelines, consult the SDS. 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. 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. The SDS for any chemical supplied by is available to you free 24 hours a day. To obtain SDSs: 1. Go to 2. In the Search field of the SDS Search page: a. Type in the chemical name, part number, or other information that you expect to appear in the SDS of interest. b. Select the language of your choice. c. Click Search. 6

7 Safety 3. To view, download, or print the document of interest: a. Right-click the document title. b. Select: Open To view the document Save Target As To download a PDF version of the document to a destination that you choose Print Target To print the document 4. To have a copy of an SDS sent by fax or , in the Search Results page: a. Select Fax or below the document title. b. Click RETRIEVE DOCUMENTS at the end of the document list. c. Enter the required information. d. Click View/Deliver Selected Documents Now. Note: For the SDSs of chemicals not distributed by Applied Biosystems, contact the chemical manufacturer. Chemical Waste Hazards HAZARDOUS WASTE. Refer to Safety Data Sheets and local regulations for handling and disposal. CHEMICAL WASTE HAZARD. Wastes produced by instruments are potentially hazardous and can cause injury, illness, or death. CHEMICAL STORAGE HAZARD. Never collect or store waste in a glass container because of the risk of breaking or shattering. Reagent and waste bottles can crack and leak. Each waste bottle should be secured in a low-density polyethylene safety container with the cover fastened and the handles locked in the upright position. Wear appropriate eyewear, clothing, and gloves when handling reagent and waste bottles. Chemical Waste Safety Guidelines To minimize the hazards of chemical waste: Read and understand the Safety Data Sheets (SDSs) provided by the manufacturers of the chemicals in the waste container before you store, handle, or dispose of chemical waste. 7

8 Preface Provide primary and secondary waste containers. (A primary waste container holds the immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.) Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). For additional safety guidelines, consult the SDS. Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood). For additional safety guidelines, consult the SDS. Handle chemical wastes in a fume hood. After emptying the waste container, seal it with the cap provided. Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local, state/provincial, or national environmental and health regulations. Waste Disposal Biological Hazard Safety If potentially hazardous waste is generated when you operate the instrument, you must: Characterize (by analysis if necessary) the waste generated by the particular applications, reagents, and substrates used in your laboratory. Ensure the health and safety of all personnel in your laboratory. Ensure that the instrument waste is stored, transferred, transported, and disposed of according to all local, state/provincial, and/or national regulations. IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal limitations may apply. 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 8

9 Safety 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: 9

10 Preface How to Obtain Support For the latest services and support information for all locations, go to then click the link for Support. At the Support page, you can: Search through frequently asked questions (FAQs) Submit a question directly to Technical Support Order user documents, SDSs, certificates of analysis, and other related documents Download PDF documents Obtain information about customer training Download software updates and patches In addition, the Support page provides access to worldwide telephone and fax numbers to contact Technical Support and Sales facilities. For more information on methods and products for PCR and reverse transcription, refer to these documents: Document Part Number Essentials of Real Time PCR: Guide Real-Time PCR Systems: 7900HT Fast Real-Time PCR System and 7300/7500 Real-Time PCR Systems Chemistry Guide TaqMan Gene Expression Master Mix: Protocol

11 Master Mix Supplies and Procedures This section covers: Overview Good Laboratory Practices Materials and Equipment Using the High Capacity RNA-to-cDNA Master Mix Overview This overview provides information about the purpose of High Capacity RNA-to-cDNA Master Mix as well as a list of the procedures, recommendations, and examples included in this protocol. Purpose of the High Capacity RNA-to-cDNA Master Mix The High Capacity RNA-to-cDNA Master Mix (Master Mix) is a 5X concentrated master mix for simple precise two-step RT-PCR. This Master Mix provides all the components needed for first-strand synthesis, except RNA, optimized for cdna synthesis over a wide dynamic range of input RNA and with a wide variety of targets. For 'No-RT control' reactions, the Master Mix also is available with a matching buffer system that contains all components with only the reverse transcriptase not added. The Master Mix produces excellent results in both real-time and conventional RT-PCR, and is optimized to produce targets less than 1,000 bases long. The Master Mix kit converts 1 pg to 1µg of RNA per 20-µL reaction. The cdna prepared using the High Capacity RNA-to-cDNA Master Mix can be used in a variety of applications, including: Quantitative PCR Short-term or long-term (archival) storage Conversion to crna 11

12 Good Laboratory Practices About This Protocol This protocol describes: Procedures for using the kits Recommendations for using the cdna created using the kits Examples of cdna conversion performance obtained using the kits Good Laboratory Practices PCR Good Laboratory Practices PCR assays require special laboratory practices to avoid false positive amplifications (Kwok and Higuchi, 1989). The high throughput and repetition of these assays can lead to amplification of a single DNA molecule (Saiki et al., 1985; Mullis and Faloona, 1987). Wear a clean lab coat (not previously worn while handling amplified PCR products or used during sample preparation) and clean gloves when preparing samples for PCR amplification. Change gloves whenever you suspect that they are contaminated. Maintain separate areas, dedicated equipment, and supplies for: Sample preparation and PCR setup PCR amplification and post-pcr analysis Never bring amplified PCR products into the PCR setup area. Open and close all sample tubes and reaction plates carefully. Do not splash or spray PCR samples. Keep reactions and components sealed as much as possible. Use positive-displacement pipettes or aerosol-resistant pipette tips. Clean lab benches and equipment periodically with freshly diluted 10% chlorine bleach solution. 12

13 Good Laboratory Practices Bibliography Kwok, S. and Higuchi, R Avoiding false positives with PCR. Nature 339: Mullis, K.B. and Faloona, F.A Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155: Saiki, R.K., Scharf, S., Faloona,F., et al., Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:

14 Materials and Equipment Materials and Equipment Available Kits Table 1 Available kits High Capacity RNA-to-cDNA Master Mix Kit Applied Bio systems Part Number High Capacity RNA-to-cDNA Master Mix, 4 96-Well Plate High Capacity RNA-to-cDNA Master Mix, 500 reactions High Capacity RNA-to-cDNA Master Mix, 200 reactions High Capacity RNA-to-cDNA Master Mix, 50 reactions High Capacity RNA-to-cDNA Master Mix, 15 reactions High Capacity RNA-to-cDNA Master Mix with No-RT Control, 500 reactions High Capacity RNA-to-cDNA Master Mix with No-RT Control, 200 reactions High Capacity RNA-to-cDNA Master Mix with No-RT Control, 50 reactions High Capacity RNA-to-cDNA Master Mix with No-RT Control, 15 reactions High Capacity RNA-to-cDNA Master Mix Components The 5X Master Mix includes optimized concentrations of the components listed in Table 1. Table 2 High Capacity RNA-to-cDNA Master Mix components Component MgCl 2 dntps (datp, dctp, dgtp, dttp) Recombinant RNase inhibitor protein Reverse transcriptase Random primers Oligo(dT) primer and stabilizers 14

15 Materials and Equipment The 'No-RT Control' kits contain equal volumes of the complete Master Mix and a buffer with all the components except for the reverse transcriptase. Table 3 Master Mix kit component amounts Kit Name Complete Master Mix Master Mix with No-RT control High Capacity RNA-to-cDNA Master Mix, 5 96-well Plates High Capacity RNA-to-cDNA Master Mix, 500 reactions High Capacity RNA-to-cDNA Master Mix, 200 reactions High Capacity RNA-to-cDNA Master Mix, 50 reactions High Capacity RNA-to-cDNA Master Mix, 15 reactions High Capacity RNA-to-cDNA Master Mix with No-RT Control, 500 reactions High Capacity RNA-to-cDNA Master Mix with No-RT Control, 200 reactions High Capacity RNA-to-cDNA Master Mix with No-RT Control, 50 reactions High Capacity RNA-to-cDNA Master Mix with No-RT Control, 15 reactions 4 plates 4 µl/well 2 tubes 1 ml/tube 2 tubes 400 µl/tube 1 tube 200 µl 1 tube 60 µl 2 tubes 1 ml/tube 2 tubes 400 µl/tube 1 tube 200 µl 1 tube 60 µl 2 tubes 1 ml/tube 2 tubes 400 µl/tube 1 tube 200 µl 1 tube 60 µl Storage of Master Mix Store all kit components at 15 to 25 C. The Master Mix can be stored for one year at 20 C, and can be stored for longer periods at 70 C. 15

16 Materials and Equipment Equipment Required but Not Supplied t Table 4 Required equipment Equipment Source Thermal cycler (one of the following): Veriti 96- Well Thermal Cycler Veriti 96- Well Fast Thermal Cycler Centrifuge with 96-well adapter Microcentrifuge Vortexer (PN ) (PN ) Major laboratory supplier (MLS) MLS MLS Materials Required but Not Supplied Table 5 lists other laboratory materials required to use the High Capacity RNA-to-cDNA Master Mix. Table 5 Required materials Materials/Consumables MicroAmp Fast 96-Well Reaction Plate MicroAmp Fast 96-Well Reaction Plate with Bar Code MicroAmp Optical 96-Well Reaction Plate MicroAmp 8-tube strip MicroAmp Fast 8-tube strip MicroAmp 8-cap strip Clear adhesive film Optical adhesive film Source (PN ) (PN ) (PN N ) (PN N ) (PN ) (PN N ) (PN ) (PN ) 16

17 Materials and Equipment Table 5 Required materials (continued) Materials/Consumables (continued) MicroAmp Optical 96-Well Reaction Plates and Optical Caps MicroAmp Optical Caps, 8 Caps/Strip Reagent Tubes with Caps, 10-mL Nuclease-free H 2 O Pipette tips, aerosol-resistant Pipettors, positive-displacement Disposable gloves Cap Installing Tool Adhesive Seal Applicator Source (PN ) (PN ) (PN ) MLS MLS MLS MLS (PN ) (PN ) 17

18 Using the High Capacity RNA-to-cDNA Master Mix Using the High Capacity RNA-to-cDNA Master Mix Overview Diagram To synthesize single-stranded cdna from RNA using the High Capacity RNA-to-cDNA Master Mix, refer to the diagram below. Add total RNA and water to the 5 RNA-to-cDNA Master Mix to create a 1 mix Perform reverse transcription in a thermal cycler Use the reverse transcription reactions (cdna) directly for quantitative or other PCR applications Store the reverse transcription reactions (cdna) Optimizing the RNA Template Amount of RNA For optimal performance of the Master Mix, recommends using RNA that is: Free of inhibitors of reverse transcription and PCR Dissolved in PCR-compatible buffer or water Free of RNase activity Use 1 pg to 1µg of RNA per 20-µL reaction. 18

19 Using the High Capacity RNA-to-cDNA Master Mix Preparing the Reverse Transcription Reactions with High Capacity RNA-to-cDNA Master Mix Prepare the Master Mix (or No-RT Control) before preparing the reaction plate. Note: Follow the same procedure to prepare No-RT controls. To prepare the reverse transcription reactions (20 µl per reaction): 1. Place the Master Mix (or No-RT Control) components on ice, then mix and briefly centrifuge them. 2. Calculate the total volume of components needed to prepare the required number of reactions. Use the table below. Component Volume per Reaction Final Concentration Master Mix (or No-RT Control) 4.0 µl 1X RNA template up to 16 µl 1 pg - 1 µg Nuclease-free H 2 O sufficient to 20 µl Total 20.0 µl IMPORTANT! Include additional reactions in the calculations to provide excess volume for the loss that occurs during reagent transfers. 3. Prepare the reaction mix according to your calculations in step 2. CHEMICAL HAZARD. High Capacity RNA-to-cDNA Master Mix may cause eye, skin, and respiratory tract irritation. Read the MSDS, and follow the handling instructions. Wear appropriate eyewear, clothing, and gloves. IMPORTANT! Prepare the reaction mix on ice. 4. Seal the plate or tubes. 5. Briefly centrifuge the plate or tubes to spin down the contents and to eliminate air bubbles. 19

20 Using the High Capacity RNA-to-cDNA Master Mix To prepare the reverse transcription reactions (20 µl per reaction): (continued) 6. Place the plate or tubes on ice until you are ready to load the thermal cycler. Performing Reverse Transcription with High Capacity RNA-to-cDNA Master Mix To perform reverse transcription: 1. Program the thermal cycler conditions as shown below, using one of the thermal cyclers listed in Table 4 on page 16. IMPORTANT! These conditions are optimized for the High Capacity RNA-to-cDNA Master Mix. Step 1 Step 2 Step 3 Step 4 Temperature ( C) Time 5 min 30 min 5 min 2. Set the reaction volume to 20 µl. 3. Load the reaction plates or tubes into the thermal cycler. 4. Start the reverse transcription run. Perform PCR amplification with one-tenth of the first-strand reaction. 20

21 Using the High Capacity RNA-to-cDNA Master Mix Storing cdna Reverse Transcription Reactions You can store cdna RT plates or tubes prepared using the High Capacity RNA-to-cDNA Master Mix for short-term or long-term storage at the temperatures shown below. IMPORTANT! Repeated freeze-thaw cycles can cause nucleic acid degradation. IMPORTANT! If required, briefly centrifuge the archive plates or tubes before storing to spin down the contents and to eliminate any air bubbles. IMPORTANT! Verify that wells or tubes do not contain air bubbles and contents are homogenous. If necessary, briefly centrifuge the plates or tubes before storage. Storage Duration Short-term (up to 24 hours before use) Long-term (up to 1 year) Storage Temperature ( C) 20 C 70 C For prolonged storage at 2 to 6 C, add EDTA to a final concentration of 1 mm to chelate cations and to prevent nucleic acid degradation. 21

22 Using the High Capacity RNA-to-cDNA Master Mix 22

23 Appendix A: Examples of cdna Yields from Reverse Transcription Quantitative PCR To determine the yield of the cdna from the reverse transcription of RNA, use quantitative PCR to test various input amounts of RNA for the cdna yield of different gene targets. The table below lists some targets and kits that you can use to evaluate the yield of cdna conversion. Table 1 Targets and kits for evaluation of cdna conversion yield Gene Target 18S GAPDH GAPDH β-actin Kit TaqMan Ribosomal RNA Control Reagents TaqMan GAPDH Control Reagents [Human] TaqMan Rodent GAPDH Control Reagents TaqMan β-actin Detection Reagents Part Number You can use other TaqMan Gene Expression Assays to evaluate the yield of cdna conversion. For a list of available assays, visit Yields for Different Targets For the example in this appendix, the input RNA was obtained from HeLa cells, and the RNA was converted to cdna using the High Capacity RNA-to-cDNA Master Mix. Figure 1 on page 24 shows an example of quantitative PCR results from the cdna for 4 different gene targets, which vary in expression levels. 23

24 Appendix A Examples of cdna Yields from Reverse Transcription There is one nonlinear response at very high input RNA amounts for the 18S gene because the real-time response is attenuated due to the C t value resulting in the baseline region. The RNA to cdna conversion is still linear at this input RNA amount. The threshold cycle (C T ) values are plotted against RNA input amounts of 1 pg to 1 µg in 20 µl reactions. Real-time PCR was performed using standard 50 µl reaction volume with 2 µl of first strand cdna as template. Figure 1 The expected ΔC T values of 3.3 for each tenfold increase in the RNA input quantity are obtained for 4 different RNA transcripts converted to cdna from different input quantities of RNA. 24

25 Index A air bubbles 21 Technical Support 10 available assays 23 B Bibliography 13 Biological Hazard Safety 8 C Chemical Hazard Warning 5 Chemical Safety 6 Chemical Waste Hazards 7 controls, No-RT 19 customer training 10 D documents, PCR and reverse transcription 10 Download PDF documents 10 Download software updates and patches 10 E equipment, required 16 F FAQs 10 G Good Laboratory Practices, PCR 12 H High-Capacity RNA-to-cDNA Master Mix storing 15 High-Capacity RNA-to-cDNA Master Mix Kits available 14 M materials, required 16 N nucleic acid degradation 21 O Obtaining SDSs 6 Overview diagram 18 P PCR good laboratory practices 12 performing reverse transcription 20 preparing reactions 19 prolonged storage 21 Protocol 12 Q quantitative PCR results 23 R required equipment 16 materials 16 reverse transcription reactions storing 21 reverse transcription reactions, performing 20 reverse transcription reactions, preparing 19 RNA template, optimizing 18 25

26 Index RNA, total input amount 18 RNA-to-cDNA reactions preparing 19 S Safety Alert Words 5 Safety Data Sheets 6 Services and support information 10 storing Master Mix 15 Synthesize single-stranded cdna from RNA 18 T TaqMan b-actin Detection Reagents 23 TaqMan GAPDH Control Reagents (Human) 23 TaqMan Ribosomal RNA Control Reagents 23 TaqMan Rodent GAPDH Control Reagents 23 Thermal cycler 16 U Using the High Capacity RNA-to-cDNA Master Mix 18 W waste disposal 8 26

27 Part Number Rev. C 06/2010 Headquarters 5791 Van Allen Way Carlsbad, CA USA Phone Technical Resources and Support For the latest technical resources and support information for all locations, please refer to our Web site at