Real-time PCR
Quantitative PCR (Q-PCR or qPCR) also known as real-time PCR is used to quantify starting amounts of nucleic acid template by analysing the amount of DNA produced during each cycle of PCR. The techniques include SYBR Green quantitative PCR, probe-based quantitative PCR and quantitative reverse transcriptase PCR (QRT-PCR).
Principle
Amplification of DNA is exponential in the early and middle cycles of a PCR (i.e. it linear on a logarithmic scale). This property can be exploited to infer the starting amount of PCR template (see diagram in Hunt tutorial).
Primer selection
For example, with the excellent free Primer3 tool on the web (v0.3, Primer3Plus). Apply the following criteria:
- pair towards 3' end (often more specific, some cDNAs don't contain)
- pair separated by an exon-exon boundary (reduces genomic background) e.g. last exon & penultimate
- amplified region must be no biger than 200 bp; usally 60-150 bp
- GC content: 50-60%
- min length: 18, max length 24 (best: 20 nt)
- melting temperature: min 60, max 63, best 60
- max Tm difference: 10 (shouldn't be more than 1 in final pair)
- max 3' self complementary: 1
- max poly-w: 3
Verify by blasting the primers sequences. Target gene should come out with the lowerst E value. No other gene should be close. Also check whether possible isoforms will be detected by the candidate primer pair.
See also: Designing primers
PCR
See also: PCR
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
Common reference mRNAs:
- glyceraldehyde-3-phosphate dehydrogenase (common metabolic enzyme)
- β-actin (common cytoskeletal enzyme)
- ribosomal proteins (e.g. RPLP0) and RNAs (28S or 18S)
- cyclophilin mRNA
- MHC I (major histocompatibility complex I)
See also
- excellent, detailed Q-PCR tutorial by Margaret and Richard Hunt, Uni of South Carolina
- Wikipedia entry on Q-PCR
