RT-PCR Troubles.. - (Nov/24/2006 )
Hi Guys,
I'm trying to do RT-PCR from the total RNA that I've. The concentration of RNA is 200ng/ul. The gene which I want to amplify is about 966 bps. I've tried with different primers sets (18 mers, 22 mers, 24 mers, 26 mers, and 32 mers) with different annealing temperature conditions (from 45 degrees C to 72 degrees C). I'm using BD biosciences One-Step RT-PCR kit. TaqMan assay shows high expression of this gene, in the cell lines that we've tested.
Can anybody suggest whats wrong with my method.. I'll be grateful and I'd like to discuss more if anyone can suggest some good tips..
I'm 
and dont know what else to do.....
Thanks in advance
I would try to switch to two step RT-PCR first of all... It would be a lot easier for you to identify the problem (is it at the RT stage, or is it at the PCR stage?). One-step might look faster and more convenient, but they are so much hassle to optimize that quite often they are not worth the attempt.
If it works with Taqman, what are the main differences in both protocols? Are both using the same primers at the RT step? Have you tried the Taqman primers in the one-step RTPCR, without the probe?
If it works with Taqman, what are the main differences in both protocols? Are both using the same primers at the RT step? Have you tried the Taqman primers in the one-step RTPCR, without the probe?
Thank you erica for your reply. I think I'd better use two-step PCR method, now.
Well, I didnt try the TaqMan primers in the one-step PCR, at all. The primers are different for both the procedures..
I've used Takara kit for the One-Step PCR... I'm a kind of dejected at this time, as I donot have any clue!! I hope I'll see some results with the two-step approach..
Thank you once again..
i think u need to check if ur RNA has been converted to cDNA first by using a constitutive gene primer when u are sure of this then go for the GSP primers which will then help u to standerdize this PCR. also make seperate aliquots of the cDNA and store at -20 and use only the dilutions of this (as the dilutions go bad after some time ie. very soon; in the sense if u use 1ul of fresh dil cDNA the after 3-4 days u require 2-3ul to get the same result). donno but the thing simply goes offf. hope this will help u.
gud luk