I have optimized the cycling condition using 2 steps Quantifast SYBR Green from Qiagen but when I try out one step Quantifast SYBR Green, I could not get the desired PCR product. In 2 steps real time PCR, I used MMLV and random primers to reverse transcribe the RNA into cDNA. In 1 step qRT-PCR, I used gene specific primers pair for cDNA synthesis and PCR in one tube (which I supposed there is no mistake here). I did an annealing temperature optimization in 1 step but also can't get the desired PCR products. I would like to know if it is possible to get expression of the target gene in my sample if it is very small, and can I use 2 steps in real time PCR?
Note: For both step 1 and 2 in Real Time PCR, total RNA is 50ng in final volume of 20ul per reaction.
I think you should verify that your gene specific primers actually amplify something in a regular RT reaction that you would run on a gel. It sounds to me that the problem are the primers.
If I am not mistaken, you used two different primer set in the one and the two steps reaction, respectively. I would suggest you try and explore the following:
(1) Use gene specific primers, but with two step method,
(2) Use random primer, but with one step method.
(3) Use primers for possitive control (internal control) for both one and two steps process
If you have positive results for no. 3, then your pcr kit ok, but if negative - you may have bad product (i.e. almost expired?).
If no. 2 is positive, your one step kit is Ok, and you may have problem with your primer, especially if no. 1 is also negative, then most probably the culprit is your primer
if no. 2 is negative and no. 1 is positive, then you may have problem with your kit
and so on, you can diagnose with other possibilities as well.
However, it might be cheaper to supect that the primers are bad and order or design new one, as it was suggested previously Aurelie Desgardin... Best of luck.
Thanks everyone. I have tried using specific primers for cDNA synthesis and there is no desired band. I used random primers for cDNA synthesis then. I can't get the desired products with conventional PCR. By using touch-down PCR, I am able to get my desired products with annealing temperature 48 deg Celcius. Thus I use the same TD-PCR protocol for fast cycling protocol using 2 steps Qiagen SYBR Quantifast PCR kit, I am able to get desired band but there are 2 faint unspecific bands. When I use the same cycling protocol using 1 step Qiagen SYBR Quantifast RT-PCR kit, I am not able to get the desired result. Is it the primers design problem?
- Badges
- Science topic