Hi Marwa,

Does your antisense transcript have a poly(A) tail? Then you can consider changing your RT primer to oligo(dT), since random hexamer usually generates short cDNA fragments and therefore may not be suitable for obtaining large intact cDNA.

Depending on your experimental purpose (e.g. if you want to subclone your gene into an expression vector or something like that), you can also consider other sources for cDNA synthesis in which your desired transcript is more abunduntly expressed than your current source. When I'm trying to amplify genes whose expression is not ubiquitous across cell types and tissues, I simply prepare a mixture of cDNAs from diverse sources (e.g. human tissue panel from Clontech) and use it as a PCR template.

If your continued efforts end up in failure but still you want to get the gene in your hands, I'd recommend gene synthesis as a last resort. Current gene synthesis technologies can handle 3kb fragment without much difficulty, you'll be provided your gene fragment with accurate sequences at reasonable prices.

Hello Marwa,

if your rna is good, cDNA probably worked. You can run your cDNA on agarose gel and see how it looks like. Also, hae you tested your primers for antisense Strand ? You can test them and you might change some paramaters in your pcr reaction.

hope it helps ...

@Dooyoung Lee Thank you so much for your kind reply. My RNA has no poly A tail and for the better quality cDNA I am trying the Thermofisher superscript IV reverse transcriptase enzyme rather than MMLV with optimal condition. I designed my Primers by using the Primer 3 software and wondering it could also be a problem with primers. Any thoughts how could I test my primers other than Gradient PCR?