will mRNA from different cell line lead to loss of expected product in RT PCR?
Posted 17 April 2013 - 09:19 PM
I have set of primers designed for Rat mRNA and I have previously used these primers in RT PCR with mRNA isolated from RIN cells and got the amplicon of expected size. Now I am repeating the same with mRNA isolated from INS 1E and I am not able to get the expected product in RT PCR, I even optimized the annealing temperature. I am getting a non specific product of different size if the annealing temperature is changed, but no band is seen while using the previously optimized annealing temperature. I can not decipher what is just happening. Will the change in template source affect the RT PCR? both are rat cell lines only.
Please someone help me in this.
Posted 17 April 2013 - 10:02 PM
- Indhumathi likes this
Posted 17 April 2013 - 11:19 PM
I have checked the isoforms, the sizes are same for all isoforms. may be i should check the splicing. but alternative splicing wont differ within species right?