Instruction Manual

SuperScript™ First-Strand Synthesis

System for RT-PCR

Catalog No. 11904-018

Version E
11 August 2003
53034
ii
Table of Contents

Kit Contents and Storage .......................................................................... 1

Introduction .............................................................................................. 2
First-Strand cDNA Synthesis ................................................................... 5
Amplification of the Target cDNA........................................................... 8
Troubleshooting...................................................................................... 11
Troubleshooting with the Control RNA ................................................. 13
Additional Protocols ............................................................................... 16
References .............................................................................................. 20
Related Products ..................................................................................... 21
Purchaser Notification ............................................................................ 23
Technical Service ................................................................................... 25

iii
iv
Kit Contents and Storage

Kit The components of the SuperScript™ First-Strand Synthesis System

Components for RT-PCR are listed below. Components are provided in
sufficient quantities to perform a total of 50 separate reactions; each
converts from 1 ng up to 5 µg of total RNA into first-strand cDNA.
The SuperScript™ First-Strand Synthesis System for RT-PCR is
stored at –20°C.
Component Amount
Oligo(dT)12-18 (0.5 µg/µl) 50 µl
Random hexamers (50 ng/µl) 250 µl
10X RT buffer [200 mM Tris-HCl (pH 8.4), 500 mM KCl ] 1 ml
25 mM MgCl2 500 µl
0.1 M DTT 250 µl
10 mM dNTP mix (10 mM each dATP, dCTP, dGTP, dTTP) 250 µl
SuperScript™ II RT (50 units/µl) 50 µl
RNaseOUT™ Recombinant Ribonuclease Inhibitor (40 units/µl) 100 µl
E. coli RNase H (2 units/µl) 50 µl
DEPC-treated water 1.2 ml
Control RNA (50 ng/µl) 15 µl
Control Primer A (10 µM) 20 µl
Control Primer B (10 µM) 20 µl

Additional The following items are required for use with the system, but they
Materials are not included:
Required • DNA polymerase for PCR
• Programmable thermal cycler
• Autoclaved 0.2- or 0.5-ml microcentrifuge tubes or autoclaved,
thin-walled PCR tubes
• Automatic pipettes and tips capable of dispensing 1–20 µl and
20–200 µl
• Disposable gloves
• Two amplification primers specific for your target mRNA
• Microcentrifuge capable of generating a relative centrifugal
force of 14,000 × g
• 37°C, 42°C, 65°C, and 70°C water baths or heat blocks

1
Introduction

System The SuperScript™ First-Strand Synthesis System for RT-PCR is

Summary optimized to synthesize first-strand cDNA from purified poly(A)+ or total
RNA. The system can be used with as little as 1 ng or as much as 5 µg of
total RNA.
After synthesis, target cDNA can be amplified with specific primers by
PCR without intermediate organic extractions or ethanol precipitations, as
shown in the diagram below.

mRNA
AAAAAA
(primer) TTTTTT

First-strand synthesis
AAAAAA
TTTTTT

Removal of RNA

TTTTTT

First-strand cDNA ready for PCR

Continued on next page

2
Introduction, Continued

Isolation of One important factor for synthesis of full-length cDNA is the

Total RNA isolation of intact RNA. The quality of the RNA dictates the
maximum amount of sequence information that can be converted
into cDNA. Thus, it is important to optimize the isolation of RNA
and prevent introduction of RNases into the RNA preparation.
RNA can be isolated by many methods. We recommend using the
Micro-to-Midi Total RNA Purification System or TRIzol® Reagent,
which yields undegraded RNA from cultured cells or whole tissue
samples. High-quality RNA can be purified from as little as 100
cells up to 1 × 107 cells or 200 mg of tissue.
Oligo(dT)-selection for poly(A)+ RNA is typically not necessary,
although it may improve the yield of specific cDNAs. Poly(A)+
RNA selection may also reduce the likelihood of genomic DNA
contamination. Frequently, RNA preparations contain small
amounts of genomic DNA that may be amplified along with the
target cDNA. If your application requires removal of all genomic
DNA from your RNA preparation, refer to DNase I Digestion of
RNA Preparation on page 18.

First-Strand The first-strand cDNA synthesis reaction is catalyzed by

cDNA SuperScript™ II Reverse Transcriptase (RT). This enzyme has been
Synthesis engineered to reduce the RNase H activity (found in other RTs) that
degrades mRNA during the first-strand reaction.
from Total
RNA Use of an RT with reduced RNase H activity results in greater full-
length cDNA synthesis and higher yields of first-strand cDNA than
obtained with other RTs. SuperScript™ II RT has been engineered to
retain the full DNA polymerase activity found in RNase H+ M-
MLV RT. This further improves the enzyme’s ability to copy long
RNA as compared to M-MLV RT or other derivatives. Because
SuperScript™ II RT is not inhibited significantly by ribosomal and
transfer RNA, it may be used effectively to synthesize first- strand
cDNA from a total RNA preparation. The enzyme exhibits
increased thermal stability and may be used at temperatures up to
50°C.
This system has been optimized to synthesize first-strand cDNA
from varying amounts of starting material. The SuperScript™ II
concentration has been lowered and RNaseOUT™ Recombinant
RNase Inhibitor has been added to the system as part of this
optimization process. Additionally, reaction conditions have been
modified to further increase the sensitivity of the system.

Continued on next page

3
Introduction, Continued

Priming First- A first-strand cDNA synthesis reaction may be primed using three
Strand cDNA different methods. The relative specificity of each primer
Synthesis influences the amount and variety of cDNA synthesized.
1. The most nonspecific of the primers, random hexamers, are
typically used when a particular mRNA is difficult to copy in
its entirety. With this method, all RNAs in a population are
templates for first-strand cDNA synthesis, and the PCR
primers confer specificity during PCR. To maximize the size
of cDNA synthesized using random hexamers, the ratio of
primers to RNA needs to be determined empirically for each
RNA preparation.
2. A more specific (and more common) priming method uses
oligo(dT) to hybridize to 3´ poly(A) tails, which are found in
the vast majority of eukaryotic mRNAs. Since poly(A)+
RNA constitutes approximately 1% to 2% of total RNA, the
amount and complexity of cDNA is considerably less than
when random hexamers are used.
Note: Using oligo(dT) to prime first-strand synthesis rather than
random hexamers or a GSP is recommended when
performing RT-PCR of a new mRNA target. Oligo(dT)
produces an RT-PCR product more consistently than
random hexamers or GSPs.
3. The most specific priming uses a gene-specific primer (GSP)
that contains sequence information of the target cDNA. First-
strand synthesis can be primed with the PCR primer that
hybridizes nearest to the 3´ terminus of the mRNA.
However, some GSPs fail to prime cDNA synthesis even
though they function in PCR on a DNA template. If gene-
specific priming fails in RT-PCR, repeat first- strand
synthesis using oligo(dT) as the primer.

RNase H The sensitivity of PCR from cDNA can be increased (especially for
Digestion long templates) by removal of the RNA template from the
Before PCR cDNA:RNA hybrid molecule by digestion with RNase H after first-
strand synthesis. Presence of RNase H during first-strand synthesis
will degrade the template mRNA resulting in decreased full-length
cDNA synthesis and decreased yields of first-strand cDNA. The
SuperScript™ First-Strand Synthesis System introduces RNase H
activity only when it is beneficial, and thus offers a unique
procedural advantage over other methods.

4
First-Strand cDNA Synthesis

Procedure This procedure is designed to convert 1 ng to 5 µg of total RNA or

Overview 50 to 500 ng of poly (A)+ RNA into first-strand cDNA. Please
review all the protocols before using the system.
A Control RNA is included in the system to verify performance.
Use 1 µl (50 ng) of Control RNA as a template for first-strand
synthesis. Refer to Troubleshooting on page 11 for additional
information.

Denature: RNA + Primer + dNTPS

65°C for 5 min

Place on ice for at least 1 min

Anneal: Oligo (dT)12-18 or GSP Random Hexamers

Add all components except Add all components except
™ ™
1 µl of SuperScript II RT 1 µl of SuperScript II RT

Mix gently Mix gently

Centrifuge briefly Centrifuge briefly

Place tubes at 42°C for 2 min Place tubes at 25°C for 2 min

™ ™
Add 1 µl of SuperScript II RT Add 1 µl of SuperScript II RT

Mix gently Mix gently

25°C for 10 min

cDNA Synthesis:

42°C for 50 min

Terminate Reaction: 70°C for 15 min

Remove RNA: Add 1 µl of RNase H

Mix gently

37°C for 20 min

PCR or store at –20°C

Continued on next page

5
First-Strand cDNA Synthesis, Continued

If you have >5 µg of total RNA, increase reaction volumes and

amount of SuperScript™ II RT proportionally.

First-Strand 1. Mix and briefly centrifuge each component before use.

Synthesis 2. Prepare RNA/primer mixtures in sterile 0.2- or 0.5-ml tubes
Using as follows:
Oligo(dT) or a
Component Sample No RT Control Control RNA
GSP
up to 5 µg total RNA n µl n µl —
Control RNA (50 ng/µl) — — 1 µl
10 mM dNTP mix 1 µl 1 µl 1 µl
Oligo(dT)12-18 (0.5 µg/µl) 1 µl 1 µl 1 µl
or
2 µM GSP 1 µl 1 µl —
DEPC-treated water to 10 µl to 10 µl to 10 µl
3. Incubate each sample at 65°C for 5 min, then place on ice for
at least 1 min.
4. Prepare the following reaction mixture, adding each
component in the indicated order. (For n samples + 1 no RT
control + 1 Control RNA reaction, prepare the reaction mix
for n + 3 reactions.)
Component Each Rxn 4 Rxns
10X RT buffer 2 µl 8 µl
25 mM MgCl2 4 µl 16 µl
0.1 M DTT 2 µl 8 µl
RNaseOUT™ Recombinant RNase Inhibitor 1 µl 4 µl
5. Add 9 µl of reaction mixture to each RNA/primer mixture,
mix gently, and collect by brief centrifugation.
6. Incubate at 42°C for 2 min.
7. Add 1 µl (50 units) of SuperScript™ II RT to each tube except
the no RT control, mix, and incubate at 42°C for 50 min.
8. Terminate the reactions at 70°C for 15 min. Chill on ice.
9. Collect the reactions by brief centrifugation. Add 1 µl of
RNase H to each tube and incubate for 20 min at 37°C before
proceeding to Amplification of the Target DNA on page 8.

Continued on next page

6
First-Strand cDNA Synthesis, Continued

For most RT-PCR applications, 50 ng of random hexamers per 5 µg

of total RNA is adequate. Increasing hexamers to 250 ng per 5 µg
of RNA may increase yield of relatively small PCR products
(<500 bp), but may decrease the yield of longer PCR products and
full-length transcripts.

First-Strand 1. Mix and briefly centrifuge each component before use.

Synthesis 2. Prepare RNA/primer mixtures in sterile 0.5-ml tubes as
Using follows:
Random
Component Sample – RT Control + RT Control
Primers
up to 5 µg total RNA n µl n µl —
Control RNA (50 ng/µl) — — 1 µl
random hexamers (50 ng/µl) 1–5 µl 1–5 µl 1–5 µl
10 mM dNTP mix 1 µl 1 µl 1 µl
DEPC-treated water to 10 µl to 10 µl to 10 µl
3. Incubate each sample at 65°C for 5 min and incubate on ice
for at least 1 min.
4. Prepare the following reaction mixture, adding each
component in the indicated order. (For n samples + 1 no RT
control and 1 Control RNA reaction, prepare the reaction mix
for n + 3 reactions.)
Component Each Rxn 4 Rxns
10X RT buffer 2 µl 8 µl
25 mM MgCl2 4 µl 16 µl
0.1 M DTT 2 µl 8 µl
RNaseOUT Recombinant
Ribonuclease Inhibitor 1 µl 4 µl
5. Add 9 µl of reaction mixture to each RNA/primer mixture,
mix gently, and collect by brief centrifugation.
6. Incubate at 25°C for 2 min.
7. Add 1 µl (50 units) of SuperScript™ II RT to each tube except
the no RT control, mix, and incubate at 25°C for 10 min.
8. Transfer the tubes to 42°C and incubate for 50 min.
9. Terminate the reactions at 70°C for 15 min. Chill on ice.
10. Collect the reactions by brief centrifugation. Add 1 µl of
RNase H to each tube and incubate for 20 min at 37°C before
proceeding to Amplification of the Target cDNA, on page 8.

7
Amplification of the Target cDNA

Procedure The first-strand cDNA obtained in the previous procedure may be

Overview amplified directly using PCR. Use only 10% of the first-strand
reaction (2 µl) for PCR. Adding larger amounts of the first-strand
reaction may actually decrease the amount of product synthesized.
PCR can use several polymerases.

Annealing and extension conditioning are primer and template

dependent, and therefore must be determined empirically.

PCR for Use Taq DNA Polymerase or Platinum® Taq DNA Polymerase.
Targets up to Platinum® Taq DNA Polymerase provides automatic hot-start
4 kb conditions for increased specificity and sensitivity.
1. Add the following to a 0.2- or 0.5-ml thin-walled PCR tube:
Volume (µl)
Component 1 Rxn 10 Rxns
10X PCR buffer minus Mg 5 50
50 mM MgCl2 1.5 15
10 mM dNTP mix 1 10
10 µM sense primer 1 10
10 µM antisense primer 1 10
Taq DNA Polymerase (5 units/µl) or
Platinum® Taq DNA Polymerase (5 units/µl) 0.4 4
cDNA (from the first-strand reaction) 2 20
autoclaved, distilled water 38.1 381
final volume 50 500
Note: The concentration of MgCl2 listed above is approximate. For
optimal amplification, determine the optimal MgCl2
concentration for your primers and template.
2. Mix gently and overlay with silicone oil or mineral oil if the
thermal cycler lacks a heated lid.
3. Incubate at 94°C for 2 min, then perform 20–40 cycles of
PCR with optimized conditions for your sample (1 min/kb
extension at 68–72°C).
4. Analyze 10 µl of the amplified sample using agarose gel
electrophoresis.

Continued on next page

8
Amplification of the Target cDNA, Continued

Annealing and extension conditions are dependent on the primers

and template, and therefore must be determined empirically.

Maximum Platinum® Pfx DNA Polymerase possesses a proofreading 3´ to 5´

Fidelity PCR exonuclease activity and provides the highest fidelity of any DNA
for Targets polymerase for PCR.
up to 12 kb 1. Add the following to a 0.2- or 0.5-ml, thin-walled tube at
either ambient temperature or on ice:
Volume (µl)
Component 1 Rxn. 10 Rxns.
10X Pfx amplification buffer 5 50
50 mM MgSO4 1 10
10 mM dNTP mix 1.5 15
10 µM sense primer 1.5 15
10 µM antisense primer 1.5 15
Platinum® Pfx DNA Polymerase (2.5 U/µl) 0.5 5
cDNA (from the first-strand reaction) 2 20
DEPC-treated water 37 370
final volume 50 500
Note: For most targets 1.25 units is sufficient. When amplifying
longer targets, above 3 kb, use 2.5 units and increase
magnesium (to ≤1.5 mM).
Note: The concentration of MgSO4 listed above is approximate.
For optimal amplification, determine the optimal MgSO4
concentration for your primers and template.
2. Mix gently and overlay with silicone oil or mineral oil if the
thermal cycler lacks a heated lid.
3. Incubate at 94°C for 2 min, then perform 20–40 cycles of
PCR with optimized conditions for your sample (1 min/kb
extension time at 68–72°C).
4. Analyze 10 µl of the amplified sample by agarose gel
electrophoresis.

Continued on next page

9
Amplification of the Target cDNA, Continued

Annealing and extension conditioning are primer and template

dependent, and therefore must be determined empirically.

Maximum Where yield of product is more critical than high fidelity, we

Yield PCR for recommend Platinum® Taq DNA Polymerase High Fidelity.
Targets up to 1. Add the following to a 0.2- or 0.5-ml, thin-walled, PCR
12 kb tube:
Volume (µl)
Component 1 Rxn. 10 Rxns.
10X High Fidelity PCR buffer 5 50
50 mM MgSO4 2 20
10 mM dNTP mix 1 10
10 µM sense primer 1 10
10 µM antisense primer 1 10
Platinum® Taq High Fidelity (5U/µl) 0.2 2
cDNA (from first-strand reaction) 2 20
DEPC-treated water 37.8 378
final volume 50 500
Note: The concentration of MgSO4 listed above is approximate.
For optimal amplification, determine the optimal MgSO4
concentration for your primers and template.
2. Mix gently and overlay with silicone oil or mineral oil if the
thermal cycler lacks a heated lid.
3. Incubate at 94°C for 2 min, then perform 20 to 40 cycles of
PCR with optimized conditions for your sample (1 min/kb
extension time at 68°C).
4. Analyze 10 µl of the amplified sample by agarose gel
electrophoresis.

10
Troubleshooting

Problem Possible Cause Suggested Solution

No bands after Procedural error in first- Use the Control RNA to verify the efficiency of
electrophoretic strand cDNA synthesis the first-strand reaction (see the next page on
analysis of troubleshooting with the Control RNA).
amplified products
RNase contamination Add Control RNA to sample to determine if
RNase is present in the first-strand reaction.
Maintain aseptic conditions to prevent RNase
contamination.
Use RNaseOUT™ Recombinant RNase Inhibitor
in the first-strand reaction.
Polysaccharide Precipitate RNA with lithium chloride (see
coprecipitation of RNA page 19) to remove polysaccharides.
Target mRNA contains Use random hexamers instead of oligo(dT) in
strong transcriptional the first-strand reaction.
pauses
Maintain an elevated temperature after the
annealing step (see page 16, First-strand cDNA
Synthesis of Transcripts with High-GC
Content).
Increase the temperature of first-strand reaction
(up to 50°C).
Use PCR primers closer to the 3´ terminus of the
target cDNA.
Too much first strand Use no more than 1/10 of the first-strand
product was used in reaction.
PCR
GSP was used for first- Try another GSP or switch to oligo(dT). Make
strand synthesis sure the GSP is the antisense sequence.
Inhibitors of RT present Remove inhibitors by ethanol precipitation of
the mRNA preparation before the first-strand
reaction. Include a 70% (v/v) ethanol wash of
the mRNA pellet.
Note: Inhibitors of RT include sodium dodecyl
sulfate (SDS), EDTA, guanidinium salts,
formamide, sodium pyrophosphate, and
spermidine.
Test for the presence of inhibitors by mixing
1 µg of control RNA with 1 µg of sample RNA
and comparing yields of first-strand cDNA.

Continued on next page

11
Troubleshooting, Continued

Problem Possible Cause Suggested Solution

Unexpected bands Contamination by Pretreat RNA as described in DNase I Digestion
after electrophoretic genomic DNA of RNA Preparation on page 18.
analysis Design primers that anneal to sequence in exons
on both sides of an intron or exon/exon
boundary of the mRNA to of amplified allow
differentiation between amplification of cDNA
and products potential contaminating genomic
DNA.
To test if products were derived from DNA, do
the No RT control.
Nonspecific annealing Vary the annealing conditions. Use Platinum®
of primers Taq DNA Polymerase for automatic hot-start
PCR.
Optimize magnesium concentration for each
template and primer combination.
Primers formed dimers Design primers without complementary
sequences at the 3´ ends.

12
Troubleshooting with the Control RNA

Introduction The Control RNA provided with this System is an 891-bp, in vitro
transcribed RNA from the chloramphenicol acetyltransferase (CAT)
gene that has been engineered to contain a 3´ poly(A) tail.
When used with Control Primers A and B in RT-PCR, the Control
RNA will result in a 500-bp product (see page 15).

First-strand Up to 1 µg of your total RNA preparation may be added to the

cDNA following mixture to evaluate the effect of this RNA on first-strand
Synthesis synthesis and subsequent amplification.
with Control 1. Label two autoclaved 0.5-ml tubes “A” and “B”. Tube A
RNA will have an addition of radioisotope to determine the
efficiency of first-strand synthesis. An aliquot from tube B
will be used for amplification.
2. Prepare the RNA/primer mixtures in sterile 0.5-ml tubes as
follows:
Volume (µl)
Component Tube A Tube B
Control RNA 1 1
Oligo(dT) 1 1
DEPC-treated water 7 8
10 mM dNTP mix 1 1
final volume 9 10
3. Incubate each sample at 65°C for 5 min and place on ice for
at least 1 min. Collect by brief centrifugation and add the
following to each tube:
Volume (µl)
Component Tube A Tube B
10X RT buffer 2 2
25 mM MgCl2 4 4
RNaseOUT RNase Inhibitor (40 U/µl) 1 1
0.1 M DTT 2 2
4. Incubate at 42°C for 2 min.
5. Add 1 µl of [α-32P]dCTP (3,000 Ci/mmol; 10 mCi/ml) to
tube A.
6. Add 1 µl (50 units) of SuperScript™ II RT to each tube.
Final volume will be 20 µl.
7. Mix gently and collect the reaction by brief centrifugation.
8. Incubate at 42° for 50 min (continued on next page).

Continued on next page

13
Troubleshooting with the Control RNA, Continued

First-strand 9. Terminate both reactions at 70°C for 15 min. Place on ice

cDNA for 1 min.
Synthesis 10. Collect the reaction by brief centrifugation. Add 1 µl of
with Control RNase H to tube B and incubate for 20 min at 37°C. Proceed
RNA, with tube B to Amplification of the Control-Synthesized
Continued cDNA on page 15.
11. Add 80 µl of distilled water to tube A and mix gently.
12. Remove two 5-µl aliquots from tube A and spot the aliquots
onto glass fiber filters. Dry one of the filters under a heat
lamp or at room temperature. This filter will be used to
determine the specific activity of the dCTP reaction.
13. Wash the other filter three times, 5 min each time, with
50 ml of ice-cold, 10% (w/v) TCA containing 1% (w/v)
sodium pyrophosphate. Wash the filter once with 50 ml of
95% ethanol at room temperature for 2 min. Dry the filter
under a heat lamp or at room temperature. This filter will be
used to determine the yield of first-strand cDNA.
14. Count both filters in standard scintillant to determine the
amount of 32P in the reaction, as well as the amount of 32P
that was incorporated.
15. Using equation 1, determine the specific activity (SA) of the
dCTP in the first-strand reaction from the counts obtained
from the unwashed filter.
SA (cpm/pmol dCTP) = cpm/5 µl
500 pmol dCTP/5 µl
16. Using equation 2, determine the yield of cDNA from the
counts obtained from the washed filter and the specific
activity calculated from the unwashed filter:
Amount of cDNA (µg) = (cpm) × (100 µl/5 µl) × (4 pmol dNTP/pmol dCTP)
(SA) × (3,030 pmol dNTP/µg cDNA)
You can assume that the yield calculated for the labeled
cDNA in tube A is equivalent to that of the unlabeled cDNA
in tube B.
17. Following first-strand cDNA synthesis using the Control
RNA, you may wish to analyze the remaining labeled cDNA
in tube A by alkaline agarose gel electrophoresis or
denaturing PAGE.
The first-strand yield using the Control RNA should be 30–50%.
Electrophoretic analysis should yield a prominent 891-bp band
representing 25–50% of the cDNA synthesized.

Continued on next page

14
Troubleshooting with the Control RNA, Continued

Amplification Following first-strand cDNA synthesis from the Control RNA,

of the Control Primers A and B can be used in the following PCR
Control- procedure.
Synthesized Primer A (anti-sense primer): 5´–GAC ATG GAA GCC ATC ACA GAC–3´
cDNA Primer B (sense primer): 5´–AGA CCG TTC AGC TGG ATA TTA C–3´

Electrophoretic analysis of DNA products amplified using the

control primers should yield a prominent 500-bp band.
1. Perform serial dilutions with the control cDNA from tube B
with DEPC-treated or sterile, distilled water. Dilute at
1:1,000, 1:10,000, 1:100,000, and 1:1,000,000.
Note: If you assume that 1% of the total RNA is mRNA, then
50 ng of Control RNA in the first-strand reaction is
equivalent to the amount of mRNA in 5 µg of total RNA.
One microliter of the 1:1,000,000 dilution of the first-strand
reaction should contain ~5,000 molecules of cDNA.
2. For each concentration, prepare a PCR mixture. Add the
following to a 0.2-ml tube sitting on ice:
Volume (µl)
Component 1 Rxn. 10 Rxns.
DEPC-treated water 37.6 376
10X PCR buffer minus Mg 5 50
25 mM MgCl2 3 30
10 mM dNTP mix 1 10
Control primer A (10 µM) 1 10
Control primer B (10 µM) 1 10
dilution of cDNA from Control RNA 1 10
Taq DNA Polymerase (5 units/µl) 0.4 4
final volume 50 500
3. Mix the contents of the tube. Centrifuge briefly to collect the
reaction components.
4. Place reaction mixture in preheated (94°C) thermal cycler.
Do an initial denaturation step: 94°C for 2 min.
5. Perform 35 cycles of PCR:
Denature 94°C for 15 sec
Anneal 58°C for 30 sec
Extend 68°C for 1 min
Note: If you are using a slow ramping machine (e.g., PE 480),
please follow the manufacturer’s suggestions.
6. Upon completion, maintain reactions at 4°C.
7. Analyze 10 µl of the amplified sample, using agarose gel
electrophoresis and ethidium bromide staining. A prominent
500-bp band should be visible for all four concentrations.

15
Additional Protocols

First-strand High-GC content mRNAs often contain stable intrinsic secondary

cDNA structures that pose barriers to reverse transcriptase and/or primer
Synthesis of annealing. Problems with RT-PCR due to secondary structure of the
target mRNA often can be overcome by increasing the volume and
Transcripts
temperature of the RT reaction.
with High-GC
Content For templates that require cDNA synthesis temperatures above
50°C, we recommend the ThermoScript™ RT-PCR System.
ThermoScript™ RT is a genetically engineered mutant of avian
retroviral reverse transcriptase with reduced RNase H activity that
supports cDNA synthesis up to 70°C.
To avoid secondary RNA structure, shift the RNA/primer mix
directly from 65°C to 50°C and prewarm the complete 2X reaction
mix to 50°C before adding it to the primer and RNA. Using a
thermal cycler simplifies the multiple temperature shifts and can
help prevent formation of secondary structure in RNA.

Continued on next page

16
Additional Protocols, Continued

Protocol This protocol is suitable for gene-specific or oligo(dT) primers, but

not random hexamers.
1. Mix and briefly centrifuge each component before use.
2. Prepare the RNA/primer mixture in a sterile 0.5-ml tube as
follows:
Component Sample No RT Control Control RNA
1 to 5 µg total RNA n µl n µl —
Control RNA (50 ng/µl) — — 1 µl
Oligo(dT)12–18 (0.5 µg/µl) 1 µl 1 µl 1 µl
or
2 µM GSP 1 µl 1 µl 1 µl
10 mM dNTP mix 2.5 µl 2.5 µl 2.5 µl
DEPC-treated water to 25 µl to 25 µl to 25 µl
3. Incubate each sample at 65°C for 5 min and immediately
transfer to 50°C.
4. Prepare the following reaction mixture, adding each
component in the indicated order. For n samples + 1 no RT
control and 1 Control RNA reaction, prepare the reaction mix
for n + 3 reactions.
Component Each Reaction 4 Reactions
DEPC-treated water 4 µl 16 µl
10X RT buffer 5 µl 20 µl
25 mM MgCl2 10 µl 40 µl
0.1 M DTT 5 µl 20 µl
RNaseOUT™ Recombinant RNase Inhibitor 1 µl 4 µl
5. Prewarm the reaction mixture to 50°C.
6. To each sample incubating at 50°C, add 25 µl of prewarmed
reaction mixture. Add 1 µl (50 units) of SuperScript™ II RT
to each tube except the no RT control, mix gently, and
incubate at 50°C for 50 min.
7. Terminate the reactions at 70°C for 15 min. Chill on ice.
8. Collect the reactions by brief centrifugation. Add 1 µl of
RNase H to each tube and incubate for 20 min at 37°C before
proceeding to PCR (page 8).

Frequently, problems associated with RT-PCR of GC-rich cDNA

are related to PCR as well as first-strand synthesis. We recommend
using PCRx Enhancer Solution, a PCR cosolvent, to facilitate
efficient amplification of GC-rich sequences.

Continued on next page

17
Additional Protocols, Continued

DNase I If amplification products are detected from the PCR reaction in the
Digestion of absence of SuperScript™ II RT, it may be necessary to eliminate
RNA residual genomic DNA from the RNA sample. After confirming the
efficiency of the first-strand synthesis reaction with the Control
Preparation
RNA, use the following protocol to remove genomic DNA from the
total RNA preparation. Amplification Grade DNase I has been
extensively purified to remove trace ribonuclease activities
commonly associated with other “RNase-free” enzyme preparations
and does not require the addition of placental RNase inhibitor.
The following procedure requires careful pipetting of all solutions
so that the concentration of divalent metal cation (Mg2+ and Ca2+) is
controlled. Because the DNase I must be heated to 65°C to
inactivate the enzyme, the concentration of free divalent metal ions
must be low enough (<1 mM) after addition of the EDTA to
prevent chemical hydrolysis of the RNA.
1. Add the following to a 0.5-ml tube on ice:
Component Amount
total RNA 1–2 µg
10X reaction buffer [200 mM Tris-HCl (pH 8.4),
500 mM KCl, 20 mM MgCl2] 1 µl
Amplification grade DNase I 1 µl
DEPC-treated water to 10 µl
2. Incubate at room temperature for 15 min.
3. Add 1 µl of 25 mM EDTA.
4. Incubate for 15 min at 65°C to heat inactivate the DNase I,
then place on ice for 1 min. Collect the reaction by brief
centrifugation. This mixture can be directly used for reverse
transcription.

Continued on next page

18
Additional Protocols, Continued

Lithium Polysaccharides or small RNAs (tRNA and 5S RNA) that co-

Chloride precipitate with mRNA may adversely affect first-strand cDNA
Purification synthesis. Highly purified mRNA can be recovered using the
following protocol adapted from Sambrook et. al. (1).
of RNA
Preparation 1. To the RNA sample in DEPC-treated water, add 0.1 volume
8 M LiCl (RNase-free) and vortex. Incubate on ice for
2 hours.
2. Centrifuge at 13,000 × g for 30 min at 4°C.
3. Remove the supernatant, being careful not to disturb the
pellet (the pellet may be difficult to see). Dissolve the pellet
in 200 µl of DEPC-treated water by drawing the pellet in
and out of a sterilized pipet tip.
4. Repeat steps 1 through 3.
5. Add 0.1 volume of 3 M sodium acetate (pH 5.2) and
2.0 volumes of absolute (100%) ethanol (–20°C). Place the
tube at –20°C for 30 min. Centrifuge at 13,000 × g for 30
min at 4°C.
6. Remove the supernatant carefully. Overlay the pellet with
100 µl of 70% ethanol (–20°C), and centrifuge at 13,000 × g
for 10 min at 4°C. Remove the supernatant, and air-dry the
RNA pellet at room temperature.
7. Dissolve the pellet in 10–100 µl of DEPC-treated water.
8. If your starting cell or tissue sample was small (1 × 103 to
1 × 105 cells), dissolve the pellet in 10 µl of DEPC-treated
water and use this entire amount for first-strand synthesis. If
your starting sample was large (>1 × 106 cells),
spectrophotometrically determine the concentration of the
purified RNA before proceeding to page 5, First-Strand
cDNA Synthesis.

19
References

Berger, S.L. and Kimmel, A.R. (1987) Methods Enzymol 152, 316.
Bracete, A.M., Mertz, L.M., Fox, D.K. (1999) Focus® 21, 38.
Chomczynski, P. (1993) Biotechniques Vol. 15, 532.
Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156.
Compton, T. (1990) in PCR Protocols: A Guide to Methods and Applications (Innis,
M., Gelfand, D., Sninsky, J., and White, T., eds.), p. 39, Academic Press, Inc.
D’Alessio, J. M., Gruber, C. E., Cain, C., and Noon, M. C. (1990) Focus® 12, 47.
Frohman, M.A., Dush, M.K, and Martin, G.R. (1988) Proc. Nat. Acad. Sci USA 85,
8998.
Gerard, G.F. (1994) Focus® 16, 102.
Gerard, G.F., D’Alessio, J.M., and Kotewicz, M.L. (1989) Focus® 11, 66.
Gerard, G.F., Schmidt, B.J., Kotewicz, M.L., and Campbell, J.H. (1992) Focus®
14, 91.
Hu, A.W., D'Alessio, J.M., Gerard, G.F., and Kullman, J. (1991) Focus® 13, 26.
Lee, C.C. and Caskey, T. (1990) in PCR Protocols: A Guide to Methods and
Applications (Innis, M., Gelfand, D., Sninsky, J., and White, T., eds.), p. 46,
Academic Press, Inc.
Sambrook J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
Simms, D., Cizdziel, P.E., and Chomczynski, P. (1993) Focus® 15, 99.
Westfall, B., Sitaraman, K., Solus, J., Hughes, J., and Rashtchian, A. (1997) Focus®
19, 46.
Westfall, B., Sitaraman, K., Berninger, M., and Mertz, L.M. (1995) Focus® 17, 62.
Westfall, B., Sitaraman, K., Lee, J., Borman, J. and Rashtchian, A. (1999) Focus® 21,
49.
Takagi, M., Nishioka, M., Kakihara, H., Kitabayashi, M., Inoue, H., Kawakami, B.,
Oka, M., and Imanaka, T. (1997) Appl. Environ. Microbiol. 63, 4504.
Sitaraman, K., Darfler, M., and Westfall, B. (1999) Focus® 21, 10.
Nathan, M., Mertz, L., Fox, D. (1995) Focus® 17, 78.
Schwabe, W., Lee, J.E., Nathan, M., Xu, R.H., Sitaraman, K., Smith, M., Potter, R.J.,
Rosenthal, K., Rashtchian, A., Gerard, G.F. (1998) Focus® 20, 30.

20
Related Products
Product Size Cat. No.
Products for RNA Isolation:
FastTrack® 2.0 mRNA Isolation Kit 6 reactions K1593-02
Micro-FastTrack™ mRNA Isolation Kit 20 reactions K1520-02
® 100 ml 15596-026
TRIzol Reagent
200 ml 15596-018
® 100 ml 10296-010
TRIzol LS Reagent
200 ml 10296-028
Micro-to-Midi Total RNA Purification System 50 reactions 12183-018

Products for Analysis:

E-Gel® Pre-cast Agarose Gels
0.8% Starter Pak 9 gels and base G5000-08
1.2% Starter Pak 9 gels and base G5000-01
2% Starter Pak 9 gels and base G5000-02
4% Starter Pak 9 gels and base G5000-04
UltraPure™ Agarose 100 g 15510-019
500 g 15510-027
UltraPure™ Agarose 1000 100 g 10975-035
100-bp DNA Ladder 50 µg 15628-019
123-bp DNA Ladder 100 µg 15613-011
250 µg 15613-029
1-Kb Plus DNA Ladder 250 µg 10787-018
1,000 µg 10787-026
Enzymes for Amplification:
Taq DNA Polymerase, Native 100 units 18038-018
500 units 18038-042
1,500 units (3 × 500 units) 18038-067
Taq DNA Polymerase, Recombinant 100 units 10342-053
500 units 10342-020
1,500 units 10342-046
(3 × 500 units)
Platinum® Taq DNA Polymerase 100 reactions 10966-018
250 reactions 10966-026
500 reactions 10966-034
5000 reactions 10966-083
Platinum® Taq DNA Polymerase High 100 reactions 11304-011
Fidelity 500 reactions 11304-029
5,000 reactions 11304-102
Platinum® Pfx DNA Polymerase 100 reactions 11708-013
(includes PCRx Enhancer Solution) 250 reactions 11708-021
500 reactions 11708-039

Continued on next page

21
Related Products, Continued
Product Size Cat. No.
Enzymes for Amplification, continued:
eLONGase® Enzyme Mix 100 reactions 10480-010
500 reactions 10480-028
PCR SuperMix 100 reactions 10572-014
PCR SuperMix High Fidelity 100 reactions 10790-020
®
Platinum PCR SuperMix 100 reactions 11306-016
Other Modifying Enzymes:
DNase I, Amplification Grade 100 units 18068-015
RNaseOUT™ Recombinant 5,000 units 10777-019
Ribonuclease Inhibitor
Ribonuclease H 30 units 18021-014
120 units 18021-071
SuperScript™ II Reverse Transcriptase 2,000 units 18064-022
10,000 units 18064-014
4 × 10,000 units 18064-071

22
Purchaser Notification

Limited Use Label This product is optimized for use in the Polymerase Chain Reaction (PCR)
License No: 4: covered by patents owned by Roche Molecular Systems, Inc. and F.
Products for PCR Hoffmann-La Roche, Ltd. (“Roche”). No license under these patents to use
that include no the PCR process is conveyed expressly or by implication to the purchaser
rights to perform by the purchase of this product. A license to use the PCR process for
PCR certain research and development activities accompanies the purchase of
certain reagents from licensed suppliers such as Invitrogen, when used in
conjunction with an Authorized Thermal Cycler, or is available from
Applied Biosystems. Further information on purchasing licenses to practice
the PCR process may be obtained by contacting the Director of Licensing
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94404 or at Roche Molecular Systems, Inc., 1145 Atlantic Avenue,
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Limited Use The purchase of this product conveys to the buyer the non-transferable right
Label License 5: to use the purchased amount of the product and components of the product in
™
SuperScript research conducted by the buyer (whether the buyer is an academic or for-
Reverse profit entity). The buyer cannot sell or otherwise transfer (a) this product (b)
Transcriptase its components or (c) materials made using this product or its components to
a third party or otherwise use this product or its components or materials
made using this product or its components for Commercial Purposes. The
buyer may transfer information or materials made through the use of this
product to a scientific collaborator, provided that such transfer is not for any
Commercial Purpose, and that such collaborator agrees in writing (a) not to
transfer such materials to any third party, and (b) to use such transferred
materials and/or information solely for research and not for Commercial
Purposes. Commercial Purposes means any activity by a party for
consideration and may include, but is not limited to: (1) use of the product or
its components in manufacturing; (2) use of the product or its components to
provide a service, information, or data; (3) use of the product or its
components for therapeutic, diagnostic or prophylactic purposes; or (4)
resale of the product or its components, whether or not such product or its
components are resold for use in research. Invitrogen Corporation will not
assert a claim against the buyer of infringement of patents owned by
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of a therapeutic, clinical diagnostic, vaccine or prophylactic product
developed in research by the buyer in which this product or its components
was employed, provided that neither this product nor any of its components
was used in the manufacture of such product. If the purchaser is not willing
to accept the limitations of this limited use statement, Invitrogen is willing to
accept return of the product with a full refund. For information on
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Licensing Department, Invitrogen Corporation, 1600 Faraday Avenue,
Carlsbad, California 92008. Phone (760) 603-7200. Fax (760) 602-6500.

Continued on next page

23
Purchaser Notification, Continued

Limited Use This product is the subject of U.S. Patent No. 5,965,399 owned by
Label License Invitrogen Corporation. The purchase of this product conveys to the buyer
No: 18: the non-transferable right to use the purchased amount of the product and
™
RNaseOUT components of the product in research conducted by the buyer (whether the
Ribonuclease buyer is an academic or for-profit entity). The buyer cannot sell or otherwise
Inhibitor transfer (a) this product (b) its components or (c) materials made using this
product or its components to a third party or otherwise use this product or its
components or materials made using this product or its components for
Commercial Purposes. The buyer may transfer information or materials
made through the use of this product to a scientific collaborator, provided
that such transfer is not for any Commercial Purpose, and that such
collaborator agrees in writing (a) to not transfer such materials to any third
party, and (b) to use such transferred materials and/or information solely for
research and not for Commercial Purposes. Commercial Purposes means any
activity by a party for consideration and may include, but is not limited to:
(1) use of the product or its components in manufacturing; (2) use of the
product or its components to provide a service, information, or data; (3) use
of the product or its components for therapeutic, diagnostic or prophylactic
purposes; or (4) resale of the product or its components, whether or not such
product or its components are resold for use in research. Invitrogen
Corporation will not assert a claim against the buyer of infringement of the
above patents based upon the manufacture, use or sale of a therapeutic,
clinical diagnostic, vaccine or prophylactic product developed in research by
the buyer in which this product or its components was employed, provided
that neither this product nor any of its components was used in the
manufacture of such product. If the purchaser is not willing to accept the
limitations of this limited use statement, Invitrogen is willing to accept
return of the product with a full refund. For information on purchasing a
license to this product for purposes other than research, contact Licensing
Department, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad,
California 92008. Phone (760) 603-7200. Fax (760) 602-6500.

Other TRIzol® is a registered trademark of Molecular Research Center, Inc.

Trademarks

24
Technical Service

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Technical Service, Continued

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26
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