cDNA Synthesis from Transcripts with High Secondary Structure Using Thermo-X™ Reverse Transcriptase

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Introduction

Thermo-X™ Reverse Transcriptase (RT) is a highly thermostable enzyme that provides full-length, first-strand cDNA synthesis at temperatures up to 70°C. The enzyme is purified from E. coli containing genes from a proprietary thermophilic eubacteria. Thermo-X™ RT has a half-life of 120 minutes at 65°C, and can synthesize cDNA from targets up to 12 kb in length.

Materials

Materials Required

  • Total RNA or mRNA
  • Random hexamers or oligo (dT) primers
  • Nucleotide Mix
  • Thermo-X™ RT and 5X Thermo-X™ RT Buffer*  
  • DNase/RNase-free water
  • RNaseOUT™ RNase Inhibitor (optional)

*Recommended Reverse Transcriptase
for difficult templates

Component 25 reactions 100 reactions
Thermo-X™ RT (200 U/µl) 25 µl 100 µl
5X Thermo-X™ RT Buffer 100 µl 4 x 100 µl
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First-Strand cDNA Synthesis

The following 20-µl reaction volume can be used for 1 pg–5 µg of total RNA or 1 pg–500 ng of mRNA.
 
  1. Add the following components to a nuclease-free microcentrifuge tube:

    2. Component Volume
    1 pg–5 µg total RNA or 1 pg–500 ng mRNA x µl
    50 ng random hexamers; or 100 µM oligo(dT)25; or 50 µM oligo(dT)20; or 2 µM gene-specific primer 1 µl
    10 mM dNTP mix (at neutral pH) 1 µl
    DNase/RNase-free water to 10 µl

     
  2. Heat mixture to 65°C for 5 minutes and cool on ice for at least 1 minute.

  3. Collect the contents of the tube by brief centrifugation and add:

    Component Volume
    5X Thermo-X™ RT Buffer 4 µl
    Thermo-X™ RT (200 units/µl) 1 µl
     Optional: RNaseOUT™ (40 units/µl) 1 µl
    DNase/RNase-free water to 20 µl

     

  4. Vortex the tube to mix and collect the contents by brief centrifugation. If using random primers, incubate tube at 25°C for 5–10 minutes

  5. Incubate at 50–70°C for 30 minutes. Use 60–64°C as a general starting point.

  6. If you are using an oligo(dT) primer, add EDTA to the reaction at a final concentration of 5 mM. For example, add 2 µl of 50 mM EDTA to a 20 µl reaction for a final concentration of 5 mM EDTA.

  7. Inactivate the reaction by heating at 90°C for 5 minutes.

  8. If you are using an oligo(dT) primer, dilute the final cDNA product 1:10 with DNase/RNase-free water before proceeding to PCR, as described in the guidelines above.

    9.  
    Store the cDNA synthesis reaction at -20°C, or proceed to PCR

Guidelines and Recommendations

Before proceeding with cDNA synthesis, note the following guidelines and recommendations:
 
We recommend using Thermo-X™ Reverse Transcriptase with random hexamers for overall best results, especially with longer targets (>5 kb).

  • Thermo-X™ can be used with oligo(dT)25 or oligo(dT)20 at all temperatures. We do not recommend using oligo(dT)12–18 at temperatures above 55°C. If you do not obtain the desired results with a shorter oligo(dT), we suggest repeating the experiment with a longer oligo(dT).
  • When using less than 50 ng of starting RNA, the addition of RNaseOUT™ is essential.
  • Thermo-X™ can be used over a broad temperature range (50–70°C). We recommend 60–64°C as a general starting point.
  • Use 2 µl (10%) or less of the first-strand cDNA product in PCR.
  • If you are using an oligo(dT) primer, you must add EDTA at a final concentration of 5 mM to the reaction prior to the final incubation step at 90°C (see the protocol on the following page). You must also dilute the final cDNA product 1:10 with water before proceeding to PCR. For example, if you are using 2 µl of the cDNA reaction in PCR, first dilute 1 µl of the cDNA product in 9 µl of water and then use 2 µl of the diluted cDNA in PCR. Note that a higher dilution (e.g., 1:100) may be appropriate for some targets.
  • PCR amplification of some targets >1 kb may require the removal of RNA complementary to the cDNA. To remove RNA, add 1 µl (2 units) of E. coli RNase H to the cDNA product and incubate at 37°C for 20 min.
  • 5X Thermo-X™ RT Buffer is viscous; vortex briefly before use and use care in pipetting. 

Buffer Composition

5X Thermo-X™ RT Buffer is composed of the following:
250 mM Tris-HCl (pH 8.3 at room temperature), 125 mM KCl, 25 mM MgCl2, and enhancer solutions

References

  1. Harrison, G.P., Mayo, M.S., Hunter, E., Lever, A.M. (1998) Pausing of reverse transcriptase on retroviral RNA templates is influenced by secondary structures both 5' and 3' of the catalytic site. Nucleic Acids Res. 26, 3433–42. 
  2. Wu, W., Henderson, L.E., Copeland, T.D., Gorelick, R.J., Bosche, W.J., Rein, A., Levin, J.G. (1996) Human immunodeficiency virus type 1 nucleocapsid protein reduces reverse transcriptase pausing at a secondary structure near the murine leukemia virus polypurine tract. J Virol. 70, 7132–42.
  3. Myers, T.W., and Gelfand, D.H. (1991) Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. Biochem. 30,7661–66
11150.pps      7-Dec-2004
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