generating cDNA from human brain RNA for cloning

[fluries]

Hi all,

I have extracted some RNA from human brain samples and want to generate cDNA from this. I prepared cDNA using random hexamers, oligo(dT) and gene specific primers. Next I try to amplify the genes of my interest, but do not get any product after PCR. All i see is the low molecualr weight primers or primer dimers.

For gene specific primers I used 2 ul of 1uM antisence primer in a 20 ul reaction volume and i do RT for 50 min at 55 C.

The genes of my interest range in size from about 1Kb to about 6 Kb.

Any suggestions as to what I am doing wrong or what else I can try.

Regards

[luisillo]

Did you heat you RNA template before RT? That's done in order to get rid of RNA's secondary structure (if there's any).

You can also run your RNA in a gel to make sure you do have RNA, or use an spectophotometer. I don't have much experience in this regarding RNA but DNA is read at 260 nm.

I hope this help you