Quantitative RT PCR

elader at ambion.com elader at ambion.com
Mon Oct 13 09:41:16 EST 1997


> *** Would you mind tutoring a third party ?!  What is a deletion
> construct, and what is/how do you apply an RNA competitor.  I assume
> you still have the same set of primers to both targets in the PCR?
***
>
I *hate* to type... but OK.
I.
First you have a gene whose level of expression you wish to quantitate
by
RT-PCR. Then you have to convince me that you shouldnt be doing RNAse
protection
assays (because they are easier, quicker, and accurate). Then you use
Oligo5.0
and design a pair of primers that function well in PCR for your gene.
Try to
keep the size of the amplicon small, the Tm of the primers equal, GC
content
50-60%, no secondary structure in the primers, etc.

II.
Then you design another pair of primers.... A 3' primer almost the
same as the
one you made, except with a 25A tail (costs you 50 bux). Then you
design a 5'
primer whose 5' end is your orig 5' primer and whose 3' end is 15
bases
downstream of the 5' primer so the resulting PCR product will be
smaller by
about 10% compared to the original. Then you take this product and
clone it int
a plasmid. Linearize it 50 bases past the insert, do a trace labeled
RNA
transcription rxn, run some numbers and get copies RNA/ul.

III. Now you can use this RNA by titering it into RT-PCR reactions
(either
random primed or dt primed...you added a tail remember?) Shoot for the
sample in
which the endogenous and competitor products are of euqal intensity.
To
facillitate accurate quantification, you can end label a primer and
run the
products on a PAGE UREA gel and use a mol. imager to get cpms.

If you have one message with lots of samples, it's worth the up front
effort.
Competitive RT-PCR is robust and reproducible. But...so are RPAs, and
they allow
probing single samples with multiple probes.

Eric

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