Priming 1st strand synthesis
General:
Oligo dT, anchored dT (3’ bias, library construction, 3’ RACE)
Random Primers (non biased distribution)
Gene Specific Primers (more sensitivity?)
Specialized:
Allele specific primers
Functionalized primers (e.g. T7 Promoter + dT, aRNA)
Primers with restriction sites on the ends (cloning)
Efficiency of different 1st strand primers on standard RT reactions
42℃ MMLV RT, G3PDH Taqman PCR
•random decamers
•oligo dT
•G3PDH primers
•no primers !!
Minimizing endogenous priming in RT Reactions
•MMLV 42℃
•SSII 48℃
•AMV 52℃
For standard qRT-PCR, random priming at 42oC is fine.
If there is any reason that you need specific priming:
• a specific site (gene specific or allele specific priming)
• the 3’ end (for 3’ RACE or other ‘anchored’ applications)
• using a chimeric or bifunctional primer (T7, restriction site)
RT inhibition of real-time PCR
…a high stringency RT reaction may be critical.
•No RT
•AMV (2 U)
•MMLV (20 U)
•SSII (20 U)
Reverse transcriptase (1/10th the amount in a standard RT reaction) added directly into a 25 ul G3PDH PCR
RT inhibition of real-time PCR:can be relieved by heat-denaturation
MMLV (20 U)
RT inhibition of real-time PCR
Heat inactivation must be prior to addition of Taq
RT inhibition of real-time PCR:can be relieved by addition of carrier protein
