4.7.2. One-Step RT-qPCR

The following is a robust, standard One-Step RT-qPCR protocol for amplifying and quantifying targets <400bp in length, using SYBR-green detection chemistry, and starting with an RNA template:

  1. Mix:
    3 μl        5 ng/ μl RNA,
    0.4 µl     10 μM Forward primer               (0.2 μM final concentration),
    0.4 µl     10 μM Forward primer               (0.2 μM final concentration),
    0.4 µl*    10 mM dNTP*                          (0.2 mM final concentration*),
    x µl        OneStep Buffer + SYBR-green  (as per manufacturer),
    y µl        nuclease-free water,
    r μl         reverse transcriptase                (as per manufacturer),
    z µl        Taq polymerase                        (as per manufacturer),
    20 µl  total volume.
    (* dNTPs are often included in the optimized buffer)
  2. Incubate in real-time thermocycler:
             95°C 5 min,
             35 cycles of:
    95°C 10 sec,
    58°C 30 sec *read for qPCR.
  3. For Melting Curve analysis of the products, incubate:
    95°C 1 min,
    55°C 1 min,
    +0.5°C increments for 5 sec, with reads from 55oC to 95oC.
    In addition, DNA sequencing of the amplified products is recommended.