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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.
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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.
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Safety
Safety Alert Four safety alert words appear in Applied Biosystems user
Words 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.
About MSDSs 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.
Waste Disposal 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.
Overview
Purpose of the The High Capacity RNA-to-cDNA Kit contains all the reagents
High Capacity needed for reverse transcription (RT) of total RNA to single-stranded
RNA-to-cDNA Kit cDNA using a reaction size of 20 µL.
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 β-globin genomic sequences and restriction site
analysis for diagnosis of sickle cell anemia. Science 230:1350-1354.
Quantity in
Component
50 reaction kit
Kit Storage Store all kit components at –15 to –25 °C (refer to real-time
protocol).
Note: RT Buffer Mix can be stored at 2 to 8 °C for up to 6 months.
Equipment and
Materials Table 2 Required equipment
Required but
Equipment Source
Not Supplied
Thermal cycler (one of the following):
• Applied Biosystems 9800 Fast Applied Biosystems
Thermal Cycler with 96-Well Aluminum (PN 4352604)
Sample Block Module
See your
• GeneAmp ® PCR System 9700
Applied Biosystems
Thermal Cycler
sales representative
Applied Biosystems
• Veriti™ 96-Well Fast Thermal Cycler
(PN 4375305)
Applied Biosystems
• Veriti™ 96-Well Thermal Cycler
(PN 4375786)
Major laboratory supplier
Centrifuge with 96-well adapter
(MLS)
Microcentrifuge MLS
Vortexer MLS
Materials/Consumables Source
Applied Biosystems
MicroAmp™ Fast 96-Well Reaction Plate
(PN 4346907)
Applied Biosystems
MicroAmp™ Optical 96-Well Reaction Plate
(PN N8010560)
Applied Biosystems
MicroAmp™ 8-tube strip
(PN N8010580)
Applied Biosystems
MicroAmp™ Fast 8-tube strip
(PN 4358203)
Applied Biosystems
MicroAmp™ 8-cap strip
(PN N8010535)
Applied Biosystems
Clear adhesive film
(PN 4306311)
Applied Biosystems
Optical adhesive film
(PN 4360954)
Applied Biosystems
MicroAmp™ Optical Caps, 8 Caps/Strip
(PN 4323032)
Applied Biosystems
Reagent Tubes with Caps, 10-mL
(PN 4305932)
Ambion
RT-PCR Grade Water
(PN AM9935)
Applied Biosystems
Cap Installing Tool
(PN 4330015)
Applied Biosystems
Adhesive Seal Applicator
(PN 4333183)
Materials/Consumables Source
Applied Biosystems
MicroAmp™ Fast 8-Tube Strip (0.1mL)
(PN 4358293)
Applied Biosystems
MicroAmp™ Optical 8-Cap Strip
(PN 4323032)
Applied Biosystems
MicroAmp™ 48-Well Optical Adhesive Film (PN 4375323)
(PN 4375928)
Applied Biosystems
MicroAmp™ Fast 48-Well Tray
(PN 4375282)
Applied Biosystems
MicroAmp™ 48-Well Base Adaptor
(PN 4375284)
Applied Biosystems
MicroAmp™ 96-Well Support Base
(PN 4379590)
+ RT Reaction - RT Reaction
or or or
C
E
D
E
C
D
and
E
F F
F
G G
H H
Preform RT reaction
Use cDNA for quantitative Store the cDNA PCR master mix between
or other real time PCR applications -15 to -25 ° C (refer to real-time protocol)
Preparing the RT Prepare the RT reaction mix using the kit components before
Reaction preparing the reaction plate.
To prepare the RT reaction mix (per 20-µL reaction):
+RT − RT
Sample up to 9 µL up to 9 µL
‡ Quantity Sufficient
IMPORTANT! Include additional reactions in the
calculations to provide excess volume for the loss that occurs
during reagent transfers.
4. Place the plate or tubes on ice until you are ready to load the
thermal cycler or Applied Biosystems Real-Time PCR
system.
Temperature (°C) 37 95 4
Storing cDNA You can store cDNA RT plates or tubes prepared using the High
Reverse Capacity RNA-to-cDNA Kit for short-term or long-term storage.
Transcription
Reactions Storage Temperature
Storage Duration
(°C)
Long-term − 15 to − 25
Quantitative PCR To determine the yield of the cDNA from the reverse transcription of
total 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 Applied Biosystems kits that
you can use to evaluate the yield of cDNA conversion.
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.
Yields for For the example in this appendix, the input total RNA was obtained
Different Targets 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 β2-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 µg in 20-µL reactions. The cDNA input is
20% of PCR reaction volume.
40
Assays
18S
35 GAPDH
ß-actin
30 ß-microglobulin
25
20
Ct
15
10
0
-1 0 1 2 3 4 5 6
Log Total RNA
Input(pg) /20 ul RT
18
Total RNA Input(pg)/20ul RT
200
16 2000
20000
200000
14
12
10
Ct
2
0 20 40 60 80 100 120 140
RT Time Minutes
30
Total RNA Input(pg)/20ul RT
200
2000
20000
200000
25
2000000
20
Ct
15
10
0 20 40 60 80 100 120 140
RT Time Minutes
20
Ct
15
10
0 20 40 60 80 100 120 140
RT Time Minutes
A H
Applied Biosystems hazards, chemical waste vii
contacting x High Capacity RNA-to-cDNA Kit 3
Technical Support x
I
B IMPORTANT, description v
biohazardous waste, handling viii
K
C Kit Components 3
CAUTION, description v
cDNA reverse transcription (RT) reactions
preparing 7
M
storing 9 materials, required 4
cDNA yields from reverse transcription, MSDSs
examples 11 description vi
chemical safety vi obtaining vi, x
chemical waste
hazards vii P
safety viii
PCR good laboratory practices 2
performing reverse transcription 8
D preparing
DANGER, description v cDNA reverse transcription (RT) reactions 7
E Q
equipment, required 3 quantitative PCR 11
G R
guidelines radioactive waste, handling viii
chemical safety vi required
chemical waste disposal vii equipment 3
chemical waste safety viii materials 4
reverse transcription, performing 8
RNA-to-cDNA Kit
storing 3
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
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03/2008