Professional Documents
Culture Documents
Protocol
Copyright 2007, Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. 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. 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. TRADEMARKS: Applera, Applied Biosystems, AB (Design), and GeneAmp are registered trademarks and MicroAmp and Veriti are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries. TaqMan is a registered trademark of Roche Molecular Systems, Inc. All other trademarks are the sole property of their respective owners.
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x
High Capacity RNA-to-cDNA Kit Protocol CONFIDENTIAL For AB Internal Use Only. Do Not Distribute.
iii
Contents
iv
High Capacity RNA-to-cDNA Kit Protocol CONFIDENTIAL For AB Internal Use Only. Do Not Distribute.
Preface
This preface covers: Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x
Safety
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 wordIMPORTANT, CAUTION, WARNING, DANGERimplies 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. Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury, illness, or death.
Preface
To minimize the hazards of chemicals: Read and understand the Material Safety Data Sheets (MSDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. (See About MSDSs on page vi.) 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 MSDS. 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 MSDS. Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturers cleanup procedures as recommended in the MSDS. Comply with all local, state/provincial, or national laws and regulations related to chemical storage, handling, and disposal. Chemical manufacturers supply current Material Safety Data Sheets (MSDSs) with shipments of hazardous chemicals to new customers. They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated. MSDSs provide the safety information you need to store, handle, transport, and dispose of the chemicals safely. Each time you receive a new MSDS packaged with a hazardous chemical, be sure to replace the appropriate MSDS in your files.
About MSDSs
Obtaining MSDSs
The MSDS for any chemical supplied by Applied Biosystems is available to you free 24 hours a day. To obtain MSDSs: 1. Go to https://docs.appliedbiosystems.com/msdssearch.html 2. In the Search field of the MSDS Search page: a. Type in the chemical name, part number, or other information that you expect to appear in the MSDS of interest. b. Select the language of your choice. c. Click Search.
vi
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 MSDS sent by fax or e-mail, in the Search Results page: a. Select Fax or Email 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 MSDSs of chemicals not distributed by Applied Biosystems, contact the chemical manufacturer.
HAZARDOUS WASTE. Refer to Material Safety Data Sheets and local regulations for handling and disposal. CHEMICAL WASTE HAZARD. Wastes produced by Applied Biosystems 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.
vii
Preface
To minimize the hazards of chemical waste: Read and understand the Material Safety Data Sheets (MSDSs) provided by the manufacturers of the chemicals in the waste container before you store, handle, or dispose of chemical waste. 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 MSDS. 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 MSDS. 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. 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.
Waste Disposal
viii
Safety
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. 017-040-00547-4; http://bmbl.od.nih.gov) Occupational Safety and Health Standards, Bloodborne Pathogens (29 CFR1910.1030; http://www.access.gpo.gov/ nara/cfr/waisidx_01/ 29cfr1910a_01.html). Your companys/institutions Biosafety Program protocols for working with/handling potentially infectious materials. Additional information about biohazard guidelines is available at: http://www.cdc.gov
ix
Preface
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
Bibliography
Kwok, S. and Higuchi, R. 1989. Avoiding false positives with PCR. Nature 339:237-238. Mullis, K.B. and Faloona, F.A. 1987. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155:335-350. Saiki, R.K., Scharf, S., Faloona,F., et al., 1985. Enzymatic amplification of -globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350-1354.
Kit Storage
Table 2
Required equipment
Equipment Source
Thermal cycler (one of the following): Applied Biosystems 9800 Fast Thermal Cycler with 96-Well Aluminum Sample Block Module GeneAmp PCR System 9700 Thermal Cycler Veriti 96-Well Fast Thermal Cycler Veriti 96-Well Thermal Cycler Centrifuge with 96-well adapter Microcentrifuge Vortexer Applied Biosystems (PN 4352604) See your Applied Biosystems sales representative Applied Biosystems (PN 4375305) Applied Biosystems (PN 4375786) Major laboratory supplier (MLS) MLS MLS
Table 3
Required materials
Materials/Consumables Source Applied Biosystems (PN 4346907) Applied Biosystems (PN 4346906) Applied Biosystems (PN N8010560) Applied Biosystems (PN N8010580) Applied Biosystems (PN 4358203) Applied Biosystems (PN N8010535) Applied Biosystems (PN 4306311) Applied Biosystems (PN 4360954) Applied Biosystems (PN 403012) Applied Biosystems (PN 4323032) Applied Biosystems (PN 4305932) Ambion (PN AM9935) MLS MLS MLS Applied Biosystems (PN 4330015) Applied Biosystems (PN 4333183)
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 MicroAmp Optical 96-Well Reaction Plates and Optical Caps MicroAmp Optical Caps, 8 Caps/Strip Reagent Tubes with Caps, 10-mL RT-PCR Grade Water Pipette tips, aerosol-resistant Pipettors, positive-displacement Disposable gloves Cap Installing Tool Adhesive Seal Applicator
Table 3
MicroAmp Fast Optical 48-Well Reaction Plate MicroAmp Fast 8-Tube Strip (0.1mL) MicroAmp Optical 8-Cap Strip MicroAmp Fast Reaction Tube with Cap (0.1mL) MicroAmp 48-Well Optical Adhesive Film
MicroAmp Fast 48-Well Tray MicroAmp 48-Well Base Adaptor MicroAmp 96-Well Support Base
+ RT Reaction
- RT Reaction
2X RT Buffer + H2O
or
C D E F G H
or
C D E F G H
or
B C D E F
384-well plate
96-well plate
Preform RT reaction
Store the cDNA PCR master mix between -15 to -25 C (refer to real-time protocol)
For optimal performance of the RNA-to-cDNA Kit, Applied Biosystems recommends using RNA that is: Free of inhibitors of reverse transcription and PCR Dissolved in TE Buffer or PCR-compatible buffer Free of RNase activity
Prepare the RT reaction mix using the kit components before preparing the reaction plate.
To prepare the RT reaction mix (per 20-L reaction):
1. Allow the kit components to thaw on ice. 2. Referring to the table below, calculate the volume of components needed to prepare the required number of reactions.
Note: Prepare the RT reaction on ice.
Component
2 RT Buffer 20 RT Enzyme Mix Nuclease-free H2O Sample Total per Reaction Quantity Sufficient
calculations to provide excess volume for the loss that occurs during reagent transfers. CHEMICAL HAZARD. 2 RT Buffer may cause eye, skin, and respiratory tract irritation. Read the MSDS, and follow the handling instructions. Wear appropriate eyewear, clothing, and gloves. 1. Mix gently and place the reaction mix on ice.
2. Seal the plates or tubes. 3. Briefly centrifuge the plate or tubes to spin down the contents and to eliminate air bubbles. 4. Place the plate or tubes on ice until you are ready to load the thermal cycler or Applied Biosystems Real-Time PCR system.
1. Program the thermal cycler conditions using one of the thermal cyclers listed in Table 2 on page 3.
IMPORTANT! These conditions are optimized for use with
2. Set the reaction volume to 20 L. 3. Load the reactions into the thermal cycler or Applied Biosystems Real-Time PCR system. 4. Start the reverse transcription run.
You can store cDNA RT plates or tubes prepared using the High Capacity RNA-to-cDNA Kit for short-term or long-term storage.
Storage Duration Short-term (up to 24 hours before use) Long-term Storage Temperature (C) 2 to 8 15 to 25
tubes before storing to spin down the contents and to eliminate any air bubbles.
10
You can use other TaqMan Gene Expression Assays to evaluate the yield of cDNA conversion. For a list of available assays, visit www.appliedbiosystems.com.
11
For the example in this appendix, the input total RNA was obtained from human Raji cells, and the RNA was converted to cDNA using the RNA-to-cDNA Kit. Figure 1 shows an example of quantitative PCR results from the cDNA for 4 different gene targets, which vary in expression levels.
Note: The amplicon for 2-microglobulin in this study was specifically designed to be A/U rich.
The threshold cycle (CT) values are plotted against log scale RNA input amounts of 1 pg to 1 g in 20-L reactions. The cDNA input is 20% of PCR reaction volume.
40 35 30 25 20 15 10 5 0
Ct
-1
3
Log Total RNA Input(pg) /20 ul RT
Figure 1 The expected CT 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 total RNA.
12 High Capacity RNA-to-cDNA Kit Protocol
To achieve optimal conversion, Applied Biosystems recommends allowing reverse transcription to occur for 60 minutes at 37 C. Figures 2, 3, and 4 show CT values plotted against reaction time in minutes for three different targets (18S, GAPDH, and 2-microglobulin) and five input amounts of RNA in 20-L reactions.
18 16 14 12 10 8 6 4 2
Ct
20
40
60
80
100
120
140
RT Time Minutes
Figure 2 The rate of conversion of 18S RNA to cDNA reaches a maximum at 90 minutes or less with 0.2 to 200 ng of input RNA (based on 20-L reactions).
13
30
25
20
Ct
15
10
20
40
60
80
100
120
140
RT Time Minutes
Figure 3 The rate of conversion of GAPDH RNA to cDNA reaches a maximum at 60 minutes or less at all RNA input levels (based on 20-L reactions).
14
30
25
20
Ct
15
10
20
40
60
80
100
120
140
RT Time Minutes
Figure 4 The rate of conversion of 2-microglobulin RNA to cDNA reaches a maximum at 60 minutes or less at all RNA input levels (based on 20-L reactions).
15
16
Index
A
Applied Biosystems contacting x Technical Support x
H
hazards, chemical waste vii High Capacity RNA-to-cDNA Kit 3
B
biohazardous waste, handling viii
I
IMPORTANT, description v
C
CAUTION, description v cDNA reverse transcription (RT) reactions preparing 7 storing 9 cDNA yields from reverse transcription, examples 11 chemical safety vi chemical waste hazards vii safety viii
K
Kit Components 3
M
materials, required 4 MSDSs description vi obtaining vi, x
P
PCR good laboratory practices 2 performing reverse transcription 8 preparing cDNA reverse transcription (RT) reactions 7
D
DANGER, description v
E
equipment, required 3
Q
quantitative PCR 11
G
guidelines chemical safety vi chemical waste disposal vii chemical waste safety viii
R
radioactive waste, handling viii required equipment 3 materials 4 reverse transcription, performing 8 RNA-to-cDNA Kit storing 3
17
Index
S
safety biological hazards ix chemical waste vii guidelines vi, vii, viii storing cDNA reverse transcription (RT) reactions 9 RNA-to-cDNA Kit 3
T
Technical Support, contacting x training, information on x
W
WARNING, description v waste disposal, guidelines viii
Y
yield of cDNA from RNA yield vs. reaction time 13 yields for different targets 12
18
Worldwide Sales and Support Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches 150 countries on six continents. For sales office locations and technical support, please call our local office or refer to our Web site at www.appliedbiosystems.com. Applera is committed to providing the worlds leading technology and information for life scientists. Applera Corporation consists of the Applied Biosystems and Celera businesses. Headquarters 850 Lincoln Centre Drive Foster City, CA 94404 USA Phone: +1 650.638.5800 Toll Free (In North America): +1 800.345.5224 Fax: +1 650.638.5884
03/2008
www.appliedbiosystems.com