Confirming successful cDNA synthesis

[neal.melvin from gmail.com]

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?