QRT-PCR

This page focuses on information specific to quantitative reverse transcriptase PCR rather than quantitative PCR in general. Typically, once cDNA is made, qPCR can either involve an endpoint PCR assay using serial dilutions of cDNA template or real-time quantitative PCR. Today real-time quantitative PCR is more common.

Protocols

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

Reference mRNAs

A mRNA used as reference or standard of a Q-PCR (and other experiments) should have the following properties:

• expressed in all cells
• constant copy number in all cells
• medium copy number for more accuracy (or similar copy number to gene of interest)

Common reference mRNAs linked to known mouse primer pairs:

• β-actin (common cytoskeletal enzyme) [1], [2]
• glyceraldehyde-3-phosphate dehydrogenase GAPDH (common metabolic enzyme) [3], [4]
• ribosomal proteins (e.g. RPLP0) and RNAs (28S or 18S)
• cyclophilin mRNA
• MHC I (major histocompatibility complex I)

• Search RTPrimerDB and check the literature before doing it from scratch.

• Check out the Eccles Lab collection of human and mouse qPCR reference genes on OWW.

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.

See also

• Q-PCR and Real-time PCR

References

1. ISBN:0415374723