9.3.4.2. Two-Step RT-qPCR

The following is a robust, standard qPCR protocol for amplifying and quantifying cDNA templates <300bp in length. The protocol is based on SYBR-green detection chemistry, with modifications for probe-based detection and qualitative PCR indicated:

1. Mix:
1.1  3 μl         cDNA           (pre-diluted 1/10, in nuclease-free water),
1.2.  0.6 µl     10 μM Forward primer      (0.3 μM final concentration),
1.3.  0.6 µl     10 μM Reverse primer      (0.3 μM final concentration),
[1.4.  0.4 µl*  10 μM TaqMan™ probe*   (0.2 μM final concentration*)],
1.5.  x µl*      TwoStep Buffer* + dNTP  (0.2 mM final dNTP),
1.6.  y µl        nuclease-free water,
1.7.  z µl        Thermostable DNA polymerase mix,    
1.8.  20 µl      total volume. 

* Use appropriate buffer for either SYBR-green or TaqMan™ probe assays. dNTP is usually included in pre-optimized buffers. If not, add separately to 0.2 mM final concentration. 

2. Incubate in real-time thermocycler:
  2.1.  5 min:95°C,  
  2.2.  35 cycles [10 sec:95°C - 30 sec:58°C - read].  
3. For SYBR-green assays, follow with Melting Curve analysis:
  3.1.  1 min:95°C,
  3.2.  1 min:55°C,
  3.3.  5 sec:0.5°C:read from 55oC to 95oC.
4. For qualitative PCR, a conventional thermocycler can be used and the products can be analysed by gel, capillary or chip-based electrophoresis.