Why are the genes which are expressed normally before lost recently(RT-PCR)?

05 May 2015 1 3K Report

I have a gene, neurotensin(NTS), expressing strongly in my cells last year but I can not amplify it by RT-PCR any more recently, all the conditions didn't change. I have positive and negative control separately so it proves that the system has no problem. I really don't know why. Can anyone have some ideas?

I usually extract RNA by:

TRIZOL

Chloroform

Isopropanol

75% Ethanol (made with DEPC water)

DEPC water to resuspend the pellet

4M Guanidium Thiocyanate

Phenol/chloroform 1:5

isopropanaol

3M NaAc

75% Ethanol (made with DEPC water)

DEPC water to resuspend the pellet

RT: M-MLV Reverse transcriptase

PCR: Thermo green buffer and dream taq

Jin Liu

Andrey, thanks! I work on the contribution of NTS and its receptor to the cancer progression. We found in some type of cancer, NTS and the receptor expressed sustainingly. I tried on several cell lines, only in one cell line I lost the expression suddenly.

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