- 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|
- Add the following components to a nuclease-free microcentrifuge tube:
- Heat mixture to 65°C for 5 minutes and cool on ice for at least 1 minute.
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
- 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
- Incubate at 50–70°C for 30 minutes. Use 60–64°C as a general starting point.
- 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.
- Inactivate the reaction by heating at 90°C for 5 minutes.
- 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.
|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|
Store the cDNA synthesis reaction at -20°C, or proceed to PCR
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.
250 mM Tris-HCl (pH 8.3 at room temperature), 125 mM KCl, 25 mM MgCl2, and enhancer solutions
- 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.
- 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.
- Myers, T.W., and Gelfand, D.H. (1991) Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. Biochem. 30,7661–66