PCR on DNA, cDNA and UHRR

[griffith]

Hi,

I have designed a set of primer from human gene within an exon. I used this set of primers to amplify in cDNA from human blood and used UHRR as a positive control, but it did not work. however, when I used human gDNA from blood, it works. I did sequencing for the PCR product, and it confirmed the sequence is the one I am expected. from the result, I am sure the primers are working, and the PCR protocol has been optmised. The question is why it does not work in human cDNA sample and UHRR?
I was thinking if the gene has low expression level in human blood, however, when I used the other primer set, it works perfectly.

Thanks for your time to read the question. any comment will be appreciated.

[jonoave]

Your descriptions is unclear.

What is the "other primer set" you are using that is working perfectly? UHRR?

So I'm assuming that the primers for your gene of interest (GOI) is not working for cDNA sample, but works on genomic DNA.

Could be due to the quality of your extracted RNA:
1. is the amplicon for your GOI located nearer to the 5'-end? 5'-ends tend to degrade faster than 3'ends. But this usually depends on the overall quality of your RNA. Do you see to 2 clear, intact bands when running your RNA on a gel?

2. similarly, what primer are you using for reverse-transcription? If you're using onl y the oligo-dT primer, there could be a reduced efficiency to amplify your GOI all the way to the 5'-end where your amplicon is located.

[griffith]

Thanks for the reply. The UHRR stands for Universal Human Reference RNA that I used as a positive control. I did run a gel for the RNA samples, they seemed fine and also the nanodrop report showed the 260/280 radtio is good. I used random primer in the reverse transcript reaction.

I have 2 sets of primers that are used to amplify the gene of interest. these 2 pair of primers (A-C and B-C) are located in 5'end (same exon). A and B are forward primer, C is the reverse one.

Pair A-C can amplify human gDNA but not in UHRR and human cDNA. however. pair B-C can amplify in human gDNA, UHRR and human blood cDNA.
from the PCR results,I assumed that there is an alternative splice variant occur in a gene deletion manner. I don't know if this is possible..
sorry for the unclear description, I hope I make it more clear this time.

[jonoave]

Thanks for clarifying your situation.

So apparently the quality of your RNA and the RT-process seems ok, so we can assume those are not a source of problem.

Since you have primer pair B-C that works on gDNA, UHRR, and cDNA; do you still need to use the primer pair A-C? What is the objective of your research?

I can't say much since we don't know what's your GOI, but I would suggest you explore a bit more into the biology.

I think that the alternative splice variant theory is possible. Tthere have been some cases where a transcribed RNA is processed differently resulting in 2 mRNAs with different lengths. Therefore it produces one protein that is shorter than the other, and both is differentially-expressed.

[griffith]

Thank you very much for the comments.
Actually my project is to detect novel alternative splice variants in my GOI.
I do PCR then cloning and sequencing. The primer pair A-C is first designed and it's not working in my cDNA. (I assumed there is a short deletion sequence in the 5'end site.) That's why primer B is designed then.