RT-PCR Protocol

Youssef Farhat, Written 10/10/11, Last updated 5/6/14

Download the PDF or Microsoft Word versions of this protocol

Introduction

Real-time Polymerase Chain Reaction (RT-PCR) is a very useful technique, but it’s subject to significant variation if not performed carefully.  I developed this protocol to reduce variation from sample to sample as much as possible.  The key is to pre-mix non-template reagents into a homogenous mixture and to pipette them after the cDNA in a manner that reduces SYBR Green spillage out of the PCR tubes.

Materials

• Reconstituted forward and reverse primers (100 µM)
• PerfeCTa SYBR Green FastMix (Quanta Biosciences, 95072-012)
• cDNA samples (1 ng/µL is the concentration I usually use)
• Molecular grade water (Invitrogen, 10977-015)
• RT-PCR Strip tubes and caps (0.1 mL) (Qiagen, 981103)
• Typical lab equipment and supplies such as micropipettors, pipette tips, and Eppendorf tubes

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Procedure

1. Thaw all reagents on ice
2. Make the F+R Primer mix
   1. Add 12 µL of the forward primer and to an empty Eppendorf tube
   2. Add 12 µL of the reverse primer to the same tube
   3. Add 976 µL of water to the tube and mix by pipetting gently
3. Make Sybr Green + Primer mix
   1. Determine number of RT-PCR reactions required for your analysis
      1. 8 reactions per standard curve
      2. 3 reactions for each sample measured
      3. Add 10% extra
   2. So, for a full plate (72 samples + 10% extra), make enough for 80 reactions
   3. For x reactions
      1. Add 10x µL Sybr Green – 800 µL for a full plate – to a new Eppendorf tube after mixing the Sybr green thoroughly with gentle pipetting
      2. Add 5x µL of the F+R primer mix – 400 µL for a full plate – to the Eppendorf with Sybr green in it and mix thoroughly with gentle pipetting (keep mixing until any swirls go away and the mixture becomes clear)
4. Plan the layout of each sample as desired (See RT-PCR Plate Layout).
5. Pipette 5 µL of cDNA into each RT-PCR tube, changing tips for each tube.
6. Pipette 15 µL of the Sybr+Primer mix into each RT-PCR strip tube, changing tips for each tube.
7. Cap and load the tubes into the RT-PCR machine.
8.  Run your RT-PCR protocol.

Helpful References

• Datasheet for SYBR Green FastMix
• RT-PCR Tutorial, by Dr. Margaret Hunt

Citing This Protocol

Farhat, Y. “RT-PCR Protocol.” The Protocol Place. July 2012. Accessed on mm/dd/yyyy. <http://protocol-place.com>