RT-PCR and basal level transcription.

George W Chacko gchacko at magnus.acs.ohio-state.edu
Sun Jan 26 15:48:38 EST 1992


How do you distinguish between basal level transcription and low 
quantities of message when RT-PCR is being used to demonstrate it's 
presence?

Scenario:

Celltype A is believed to contain message for gene I.

Celltype B is known to contain message for gene I.

Celltype B frequently contaminates (albeit at a low level) Celltype A
preparations.

Celltype A has a much lower transcription rate (overall) than Celltype B.

The message is being demonstrated by RT-PCR using gene I specific primers.

Control sets of primers believed to amplify celltype specific message are
used to evaluate contamination. This is based on the fact that the proteins
encoded by these messages are not detectable in the abovementioned cell
types.

Celltype B primers consistently amplify from Celltype A preparations
although the A:B ratio is in the region of 4000:1 -> 6000:1. As determined
by an electronic cell counter. Celltype A and B are both found in blood
and total RNA is extracted by guanidine:sarcosine lysis and acid phenol
extraction.

RT-PCR is performed by using a poly dT:random hexamer mix and 0.5-1 ug
of total RNA. PCR amplification is done with 40 cycles and 1/5 of the
RT product.

Question:

Is the type B message due to contamination or due to basal level transcription
in Celltype A?

Maybe we could keep this discussion on the net unless it gets too unwieldy.

Thanks

George



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