Confirming successful cDNA synthesis

Hello,

I am just starting reverse transcription-based PCR in a new lab (it's
the first time that PCR has been attempted here). I have been using
the Protoscript II kit from NEB, but have not seen any bands on my gel
in the end, even using the control rat liver RNA and GAPDH PCR primers
and conditions included in the kit. Incidentally, I have primed the RT
using the supplied anchored oligo dTs.

I would like to know whether the RT step or the PCR is the problem. I
can't get my hands on any other general PCR primers to other genes at
the moment, which is what I would use to confirm that the RT went
well. I have confirmed that the control total RNA included in the kit
is good, as assayed by running a denaturing RNA gel: both the 28s and
18s bands were very strong.

From what I've read, the typical way to confirm a successful RT is to
include radiolabelled nucleotides during the RT step, and then analyse
the products on a gel afterwards. Since I'd rather not resort to
radioactivity-based experiments, is there any other way that I can use
to confirm that I actually have cDNA? I did try to run a large amount
of the RT reaction on a gel itself to look for signs of cDNAs of
various lengths, but saw nothing.

I suppose one reason that I saw nothing is that the cDNA may have been
too dilute... can I use standard ethanol precipitation to precipitate
and concentrate my supposed cDNA sample?

Hi Neal,

1. The Reason why you are not able to the band in the
gel(agarose/polyac i presume) is that you are using some intercalating
agent like ethidium bromide which gives fluoresces under UV light. RNA
has sec structure hance it can take EtBr..but not cDNA it is single
stranded..you will get double stranded dna only if u run a pcr using
cDNA. Also cDNA as far as i know does not form any secondary
structures hence it cant hold any intercalating agent..and hence no
band.

2. In relation to the above paragraph, mRNAs having secondary
structure are known to repress the translation and mutation induced in
those regions can enhance gene expression. So if ethidium bromide is
intercalating in mRNAs then it is in low abundant mRNAs which probably
have secondary structure..other wise they are other types of RNAs..i
guess mRNA make up a small percentage of total RNA

Reply if it did solve on of your question.
I am waiting for the aswer to second question of yours from others.

Others can also educate me about whatever i have said.

Sudheendra.


On Jan 1, 2008 8:40 AM, <neal.melvin@gmail.com> wrote:



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