RT-PCR

[banuy]

I am using SuperScript First-Strand Synthesis System for RT-PCR. I performed the reaction with control RNA and it worked. I tried 4 different primer sets for 4 different genes. 3 of the primer sets which amplify smaller than 500 bp fragments are working but 1 primer set which should amplify a 2 kb fragment is not working. Are there anyone who faced with problems with moderately long product amplifications?

[cathbr]

Hi,

a lot of things could happen between the RNA and your PCR... If you give me more details I might be able to help
For ex:
the conditions that you are using for the PCR
if you are using specific or random primers for the RT
if you try to amplify the same products, or spliced varients,... with 4 different primers...
Are you sure that your 2kb fragments is within the cDNA that you created?
Did you try to increase the elongation time?
Did you try different annealing temperatures?

[bravebeaker]

as far as i know and correct me if i am wrong, RT-PCR amplification products should be short, not too short but not too long, if I understood correctly you are amplifying a 2Kb amplicon I think that is very very long.

[JackBean]

why should it be short?

[bravebeaker]

I am saying based on the kit I am using which is SYBR green by TOYOBO and also many qPCR protocols over the internet as well as papers indicate the use of a short amplicon between 80 to 200 bp... what do you think? maybe banuy's experiment is different?

[JackBean]

First strand synthesis and qPCR are IMHO something different. Yes, for qPCR one should have only short amplicons, but I don't see reason why should you amplify only short pieces during reverse transcription.

[bravebeaker]

Now I see your point, but in my case I use the primer mix that comes with the RT kit by Takara..

[JackBean]

random hexamers?

[bravebeaker]

yes

[JackBean]

in such case you will have mixture of random lengths and only the length of amplification step will influence the maximal length of your amplicons