QRT-PCR

See Q-PCR for much useful information.

This page focuses on information specific to quantitative reverse transcriptase PCR rather than quantitative PCR in general.

Protocols

• qRT-PCR/Two tube (in progress)
• qRT-PCR/Single tube

Notes

• The most commonly used specialist reverse transcriptase enzyme for cDNA production is AMV reverse transcriptase. It has RNase H activity (so that RNA molecules are only transcribed once) and has a high temperature stability (to reduce RNA secondary structure and nonspecific primer annealing).
• Since RNA can degrade with repeated freeze-thaw steps, experimental variability is often seen during successive reverse transcription reactions.
• Reverse transcriptase enzymes are notorious for their thermal instability. Repeated removals from the freezer can degrade the efficiency of the enzyme.
• Producing total cDNA from total RNA can be advantageous because
  1. cDNA is more stable than RNA so making total cDNA allows you to make multiple sequence-specific RNA measurements.
  2. This approach could reduce experimental variability from RNA degradation.
• To make total cDNA
  1. use a polyT primer (most but not all eukaryotic mRNA) or random decamers (prokaryotic mRNA).
  2. Random decamers give longer cDNAs on average than random hexamer primers.
  3. Use longer reverse transcription reaction times.
  4. Ensure that the concentration of deoxynucleotides doesn't run out.

References

1. ISBN:0415374723