RNA extraction and RT PCR product variability.
mckenzir at CRYPTIC.RCH.UNIMELB.EDU.AU
Tue Sep 14 22:13:54 EST 1999
I am using the guanidinium thiaocyanate RNA extraction method followed by
RT-PCR. I quantitate the RNA at 260/280 and use 1 ug in a 25 ul RT reaction
using M-MLV. 4ul of this final product after 1hour at 37C goes into a 100ul
PCR reaction. I find that I am getting huge variation between products. Two
samples can be processed side by side with both having a similar RNA
concentration and ratio (Average 1.8-1.9) and similar protein concentration
(Estimated by spectrophotometre) yet the final product can be either very
strong or non-existant. I had thought residual ethanol or
betamercaptoethanol may have been the problem but careful washing and
drying of the pellet has yeilded no positive results.
Can anyone help me?
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