Introduction

The SuperScript™ III First-Strand Synthesis System SuperMix is an optimized SuperMix formulation for first-strand cDNA synthesis from purified poly(A)+ or total RNA. RNA targets from 100 bp to >12 kb can be detected with this system. The amount of starting material can vary from 0.1 pg to 5 μg of total RNA. The kit includes SuperScript™ III/RNaseOUT™ Enzyme Mix, 2X First-Strand Reaction Mix, and Annealing Buffer. SuperScript™ III Reverse Transcriptase, included in the Enzyme Mix, is a version of M-MLV RT that has been engineered to reduce RNase   activity and provide increased thermal stability (1, 2).

The enzyme can be used to synthesize cDNA at a temperature range of 45–55°C, providing increased specificity, higher yields of cDNA, and more full-length product than other reverse transcriptases. Because SuperScript™ III RT is not significantly inhibited by ribosomal and transfer RNA, it can be used to synthesize cDNA from total RNA. RNaseOUT™ Recombinant RNase Inhibitor is included in the Enzyme Mix to safeguard against degradation of target RNA due to ribonuclease contamination.


The 2X First-Strand Reaction Mix includes 10 mM MgCl2 and 1 mM of each dNTP in a buffer formulation that has been optimized for first-strand synthesis of cDNA. The Annealing Buffer is used in the initial template-primer annealing step. Separate tubes of oligo(dT)20 and random hexamers are also provided. cDNA synthesis can be performed using either total RNA or poly(A)+-selected RNA primed with oligo(dT), random primers, or a gene-specific primer. Recommendations for PCR amplification of the first-strand cDNA are provided below.
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Materials

Kit Contents

The components of the SuperScript™ First-Strand Synthesis System 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
SuperScript™ III/RNaseOUT™ Enzyme Mix100 µl
2X First-Strand Reaction Mix (contains 10 mM MgCl2, and 1 mM each dNTP)500 µl
Annealing Buffer 50 μl
Oligo(dT)20 (50 μM)
50 µl
Random hexamers 50 µl

Quality Control

A minimum of 40 ng of a 1181-bp RT-PCR product was obtained from 10 pg of total HeLa RNA and human GAPDH primers. A minimum of 200 ng of a 5.6-kb RT-PCR product was obtained from 100 ng of total HeLa RNA and human DNCH primers
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Ordering Information

Sku Name Size Price Qty
18080400 SuperScript® III First-Strand Synthesis SuperMix 50 reactions USD 452.00
10966018 Platinum® Taq DNA Polymerase 120 reactions USD 99.00
10966026 Platinum® Taq DNA Polymerase 300 reactions USD 258.00
10966034 Platinum® Taq DNA Polymerase 600 reactions USD 459.00
11304011 Platinum® Taq DNA Polymerase High Fidelity 100 reactions USD 165.00
11304029 Platinum® Taq DNA Polymerase High Fidelity 500 reactions USD 770.00
11708013 Platinum® Pfx DNA Polymerase 100 reactions USD 131.00
11708021 Platinum® Pfx DNA Polymerase 250 reactions USD 314.00
11708039 Platinum® Pfx DNA Polymerase 500 reactions USD 546.00
15596026 TRIzol® Reagent 100 mL USD 167.00
15596018 TRIzol® Reagent 200 mL USD 299.00
18068015 DNase I, Amplification Grade 100 units USD 118.00

Protocol - First-Strand cDNA Synthesis

The following procedure is designed to convert 0.1 pg to 5 μg of total RNA or 0.1 pg to 500 ng of poly(A)+ RNA into first-strand cDNA:

  1. Mix and briefly centrifuge each component before use. Preheat the thermal cycler to 65°C.

  2. Combine the following in a 0.2-ml thin-walled PCR tube on ice:

  3. Component Amount
    up to 5 μg total RNAn μl
    Primer (50 μM oligo(dT)20, or 2 μM gene-specific primer, or 50 ng/μl random hexamers)1 μl
    Annealing Buffer1 μl
    RNase/DNase-free waterto 8 μl


  4. Incubate in a thermal cycler at 65°C for 5 minutes, and then immediately place on ice for at least 1 minute. Collect the contents of the tube by brief centrifugation.

  5. Add the following to the tube on ice: 2X First-Strand Reaction Mix 10 μl SuperScript™ III/RNaseOUT™ Enzyme Mix 2 μl

  6. Vortex the sample briefly to mix, and collect by brief centrifugation. Incubate as follows: Oligo(dT)20 or GSP primed: 50 minutes at 50°C Random hexamer primed: 5–10 minutes at 25°C, followed by 50 minutes at 50°C

  7. Terminate the reactions at 85°C for 5 minutes. Chill on ice.

Store the cDNA synthesis reaction at -20°C, or proceed directly to PCR.

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Recommendations and Guidelines

Required Materials

  • 0.2-ml, nuclease-free thin-walled PCR tubes
  • Thermal cycler, preheated to 65°C
  • Ice
  • RNase/DNase-free water
  • Microcentrifuge
  • Vortex mixer

RNA
  • High-quality, intact RNA is essential for full-length, highquality cDNA synthesis. This kit is designed for use with 0.1 pg to 5 μg of total RNA or 0.1 pg to 500 ng of poly(A)+ RNA.
  • RNaseOUT™ Recombinant RNase Inhibitor is included in the Enzyme Mix to safeguard against degradation of target RNA due to ribonuclease contamination of the RNA preparation.
  • To isolate total RNA, we recommend TRIzol® Reagent (Cat. Nos. 15596-026/-018) or the Micro-to-Midi Total RNA Purification System (Cat. no. 12183-018). Isolation of poly(A)+ RNA is typically not necessary, although it may improve the yield of specific cDNAs; we recommend the FastTrack® 2.0 mRNA Isolation Kit (Cat. no K1593-02).
  • Small amounts of genomic DNA in the RNA preparation may be amplified along with the target cDNA. If your application requires removal of all genomic DNA from your RNA preparation, we recommend using DNase I, Amplification Grade (Catalog no. 18068-015). DNase I, Amplification Grade, 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.

cDNA Synthesis Reaction Conditions

  • For difficult or high GC-content templates, use a 55°C cDNA synthesis temperature.
  • Keep all components, reaction mixes, and samples on ice. After preparation of the annealing mixture, transfer the mixture to the preheated thermal cycler.

Primers

First-strand cDNA synthesis can be primed using random hexamers, oligo(dT), or gene-specific primers (GSPs):

  • Random hexamers are the most nonspecific priming method, and are typically used for difficult or high GC-content mRNA. Using random hexamers, all RNAs in a population are templates for first-strand cDNA synthesis, and PCR primers confer specificity during PCR.
  • Note:  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 small PCR products (<500 bp), but may decrease the yield of longer PCR products and full-length transcripts.
  • Oligo(dT), a more specific priming method, is used 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 with random hexamers. We recommend using the oligo(dT)20 provided in the kit.
  • Note:  Oligo(dT) is recommended over random hexamers or GSPs for new mRNA targets. Oligo(dT) produces an RT-PCR product more consistently than random hexamers or GSPs.
  • The most specific priming method uses a gene-specific primer (GSP) for the sequence of interest. First-strand synthesis can be primed with the PCR primer that hybridizes nearest to the 3´ terminus of the mRNA. Note that some GSPs fail to prime cDNA synthesis even though they work in PCR on DNA templates. If gene-specific priming fails in RT-PCR, repeat first-strand synthesis using oligo(dT) as the primer.

Guidelines for PCR

The first-strand cDNA from the synthesis reaction may be amplified directly using PCR. We recommend using 10% of the first-strand reaction (2 μl) for PCR. However, for some targets, increasing the amount of first-strand reaction to up to 10 μl may result in increased product yield.

We recommend the following DNA polymerases:

  • Platinum® Taq DNA Polymerase provides automatic hot-start conditions for increased specificity and sensitivity. It is recommended for targets up to 4 kb.
  • Platinum® Taq DNA Polymerase High Fidelity provides increased fidelity and higher yields for targets up to 15 kb.
  • Platinum® Pfx DNA Polymerase possesses a proofreading 3´ to 5´ exonuclease activity and provides maximum fidelity for PCR. It is recommended for targets up to 12 kb.


  • Consult the product documentation provided with each DNA polymerase for recommended protocols and optimization guidelines. Documentation is also available on our Web site at www.invitrogen.com.
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Troubleshooting

Problem
Probable Solution
No or faint bands after analysis of amplified products
 
Procedural error in first-strand cDNA synthesis
Repeat the procedure, being careful to follow each step. Be careful to include the
Annealing Buffer when adding primers and template for optimal yield.
 RNase contamination
Maintain aseptic conditions to prevent RNase contamination. RNaseOUT™ is
included in the Enzyme Mix to inhibit RNases.
 Polysaccharide coprecipitation of RNA
Precipitate RNA with lithium chloride to remove polysaccharides, as described in
Sambrook et al.
Target mRNA contains strong transcriptional
pauses
  • Use random hexamers instead of oligo(dT) in the first-strand reaction.
  • Increase the reaction temperature to 55°C.
  • Prewarm the 2X First-Strand Reaction Mix to 55°C and hold the primer-template mixture at 55°C, then add the Enzyme Mix. Perform cDNA synthesis at 55°C.
  • Use PCR primers closer to the 3´ terminus of the target cDNA.
Too little first-strand product was used in PCR
Use up to 10 μl of the first-strand reaction.
 GSP was used for first-strand synthesis
Try another GSP or switch to oligo(dT). Make sure the GSP is the antisense
sequence.
Inhibitors of RT present
Remove inhibitors by ethanol precipitation of mRNA preparation before the firststrand
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.
Unexpected bands after electrophoretic analysis
 
Contamination by genomic DNA
  • Pretreat RNA with DNase I, Amplification Grade (Cat. no. 18068-015), as described
    in the DNase I documentation.
  • Design primers that anneal to sequence in exons on both sides of an intron or at the exon/exon boundary of the mRNA to differentiate between amplified cDNA and potential contaminating genomic DNA.
Nonspecific annealing of primers
  • Vary the annealing conditions. Use Platinum® 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.


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References

  1. Berger, S.L. and Kimmel, A.R. (1987) Methods Enzymol 152, 316.

  2. Bracete, A.M., Mertz, L.M., Fox, D.K. (1999) Focus® 21, 38.

  3. Chomczynski, P. (1993) Biotechniques Vol. 15, 532.

  4. Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156.

  5. 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.

  6. Frohman, M.A., Dush, M.K, and Martin, G.R. (1988) Proc. Nat. Acad. Sci USA 85, 8998.

  7. Gerard, G.F. (1994) Focus® 16, 102.

  8. Sambrook J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor.

  9. Simms, D., Cizdziel, P.E., and Chomczynski, P. (1993) Focus® 15, 99.

  10. Westfall, B., Sitaraman, K., Solus, J., Hughes, J., and Rashtchian, A. (1997) Focus® 19, 46.

  11. Westfall, B., Sitaraman, K., Lee, J., Borman, J. and Rashtchian, A. (1999) Focus® 21, 49.

  12. Takagi, M., Nishioka, M., Kakihara, H., Kitabayashi, M., Inoue, H., Kawakami, B., Oka, M., and Imanaka, T. (1997) Appl. Environ. Microbiol. 63, 4504.

  13. Sitaraman, K., Darfler, M., and Westfall, B. (1999) Focus® 21, 10.

  14. Nathan, M., Mertz, L., Fox, D. (1995) Focus® 17, 78.

  15. 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.


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18080400.pps                                 8-Oct-2004