RT-PCR - PCR conditions optimization (Jul/22/2009 )

Hi all,

Iam stuchup with one basic problem
I could succesfully amplify my gene of intrest using gene specific primers from genomic DNA but unable to do it with Ist strand cDNA template.
what parameters do I need to optimize for PCR amplification.
I have used same conditions for PCR ampliofication for both genomic DNA and cDNA amplification.

Appreciate ur suggestions

novagen on Jul 22 2009, 09:23 AM said:

Can you use more RNA template in you cDNA reactions? I have a colleague that is trying to measure expression of some genes, but they're apparently expressed in low amounts, because he had a hard time getting Ct values lower than 30. He ended up adding a lot more RNA to his cDNA reactions and was able to get his Ct values into the 20s.

Is (could) the gene you're trying to measure (be) inducible?

Also, what primer are you using for your cDNA synthesis reaction, gene specific? Hexamers? Oligo dT?

Prokaryotic or eukaryotic?

Hi

The gene I am trying is an inducible gene from a plant.
only after induction, I have isolated RNA and used sufficient template for the rxn
what else do I do.
The gene iam trying to isolate has got many introns
Do I need to change annealing temp for PCR amplification

ThanX

novagen on Jul 22 2009, 10:27 AM said:

Are you running any positive controls with a constitutively expressed gene to ensure the first strand synthesis is working? Like B-actin or rRNA?

Hi fishdoc,

I always run actin first and only then go for specific gene

Thanx a lot for the reply

novagen on Jul 22 2009, 11:31 AM said:

So, are you saying that your actin controls are all positive, but you inducible gene is negative from the same sample?

If that's the case, my first instinct is that either the primers for the cDNA synthesis are not amplifying your transcript if you're using gene specific primers. With oligo dT or hexamers, that shouldn't be a problem. The second thing would be that expression is too low to detect, even though you say you've induced it. How much RNA are you adding to your cDNA synthesis reaction? Have you tried adding more to see if you get a better signal?

Hi fishdoc,

thanx for the reply
could be the expression level is low.
but once I got the cDNA amplicon lower than 250bp which I am supposed to get at 1.4kb.what could that be?

Thank you

novagen on Jul 22 2009, 09:50 PM said:

I'm sorry, but I don't follow what you're saying there. I've always tried to keep my RT-PCR amplicons pretty small, preferably below 500 bp, but usually 100-250 bp. Not sure if your amplicon size is a problem or not, but if 1.4 kb is what you're expecting to get, that may be the reason for the low yield.

Hi fishdoc,

Today i tried to optimize the pcr conditions for RT-PCR (which I previously told about 250bp band)
I have decresed the annealing temp and increased the amount of template(Ist strand cDNA)
I could see 2bands i.e 250bp and 400bp along with primer dimers.but my doubt is do we get nonspecific amplification in RT-PCR

thanX

novagen on Jul 23 2009, 09:45 AM said:

That depends on what your primers were for cDNA synthesis. If you used oligo dT or hexamers, there's a good possibility of non-specific amplification. If you used a gene specific primer for cDNA synthesis, there's much less chance of non-specific amplification. If you're concerned about the product, you could purify it and sequence to confirm.