will mRNA from different cell line lead to loss of expected product in RT PCR? - (Apr/17/2013 )
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.
First you need to make sure your PCR works and your gene is expressed in the new cell line. If the answer to both questions is yes, then there is possibility that the two types of cells give you different sized bands, or one does not give you amplification at all due to alternative splicing. Even you don't know what is going on in the two cells, but you can look into databases such as NCBI AceView to know the structure of the gene and potential isoforms. You should also check your primer location to know which region your primers bind to. You can use the in silico PCR tool or the Splice Center to do so. After gathering all the information, you should predict whether the bands you got are non-specific or isoforms.
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?