There are two approaches for performing RT-PCR: one-step and two-step RT-PCR

Order RT-PCR Kits

One-Step RT-PCR

One-step RT-PCR combines the first-strand cDNA synthesis (reverse transcription) reaction and PCR reaction in the same tube, simplifying reaction setup and reducing the possibility of contamination. One-step RT-PCR allows easier processing of large numbers of samples, and helps minimize carryover contamination, since tubes are not opened between cDNA synthesis and amplification. By amplifying the entire cDNA sample, one-step RT-PCR can provide greater sensitivity—from as little as 0.01 pg total RNA. One-step reactions allow for the use of sequence-specific primers only.

Two-Step PCR

Two-step PCR begins with the reverse transcription of either total RNA or poly(A)+ RNA into cDNA using a reverse transcriptase. Following first-strand synthesis, the cDNA is transferred to a separate tube for the PCR step. Two-step RT-PCR is useful for detecting multiple messages from a single RNA sample. You’ll get greater flexibility when choosing primers and polymerase than with one-step RT-PCR systems. When performing two-step RT-PCR, you have the option of using either oligo(dT), random hexamer, or gene-specific primers, and then PCR is performed with either Platinum® Taq DNA Polymerase, Platinum® Taq DNA Polymerase High Fidelity, or your choice of PCR enzyme.

Comparison of One-Step & Two-Step RT-PCR Procedures

  Two-Step Procedure One-Step Procedure
Prime first-strand cDNA with:
  • Oligo(dT) primer
  • Random hexamers
  • Gene-specific primers
  • Gene-specific primers
ProvidesFlexibility
  • Choice of primer
  • Choice of amplification system
  • Ability to save some RNA sample for later use
  • Ability to optimize for difficult RT-PCR (combine with Platinum® enzymes for higher specificity or combine with Platinum® Pfx for greater fidelity)
Convenience
  • Amplifcation enzymes premixed with reverse transcriptase
  • Fewer pipetting steps and reduced chances of contamination
  • High sensitivity
Recommended uses:
  • Ideal for detection or quantifying several messages from, a single sample
  • Ideal for analysis of large numbers of samples
  • Ideal for real-time quantitative RT-PCR