Semi-quantitative RT-PCR

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Tue Jan 18 12:34:33 EST 2000


In article <8623il$k9i$1 at oyez.ccc.nottingham.ac.uk>, Samuel Gray
<samuel.gray at nottinghNOSPam.ac.uk> writes
>Can anyone offer any suggestions as to how to increase the sensitivity of a
>RT-PCR system? We are trying to perform simple semi-quantitative RT-PCR on
>muscle biopsy samples, comparing a PKC isoform mRNA with GAPDH mRNA levels,
>but however I adjust cycles, template concentrations and conditions I cannot
>detect any PCR products within the linear amplification range - only at
>plateau.

How do you know that you actually at plateau? Presumably extra cycles
give no more product? What do you actually get if you remove an aliquot
or better still take off tubes at every cycle for say 5 cycles before
the plateau. Surely you see an increase in intensity until it plateau's?

How bright is the final product on an EtBr gel. 10% of your PCr should
be a bright band if it truly is at plateau. Any false priming products
that are limiting the yield of your product i.e. primer dimers in
particular which you may not see unless you run the right gel
conditions?

What is your RT-PCR protocol? Single tube, two tube etc? Can you get a
housekeeping gene to behave 'properly' in your system, maybe (but not
only) B-actin, cyclophilin, GAPDH etc. I presume you run an internal
control primer pair, again maybe a housekeeping RNA, in with your normal
RT-PCR's just to show that nothing is inhibitory etc.  

Back to increasing sensitivity.

Nested PCR. Stop your PCR several cycles well before plateau and re-PCR
an aliquot with internal primers for however no. of cycles you
experimentally determine will give you the discrimination you require.

For quantification look at using a mimic or competimer etc.

Duncan
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk




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