RNA isolation from isolatedislets and RT-PCR - (Jul/15/2001 )
Hi friends,I was trying to do RT-PCR in isolated pancreatic islets.I have used the primers for which I got good results in the brain regions. But in the case of iselts I didn't get any bands. The problem won't be with the protocols, but with the RNA degradation while RT-PCR. Can you suggest any method to block the Rnase activity of Enzymes which we use for the RT-PCR. I am Using Human placental Rnase Inhibitor in the PCR mixture. I have tried with different concentrations of RNA then also I didn't get anything.Then I have come to a conclusion that RNA is getting degraded during the reaction. Now I am thinking to increase the concentration of inhibitor. Is it enough or you have any suggestions please help me? I am doing Ph.D in Cochin University, Kerala, India. As I have to submit my thesis by the middle of August I am in tension. So please help to get good results
As I understand you are performing RT with specific primers which you have proved effective for brain tissue. Are you sure that they are suitable for the RNA isolated from pancreatic islets?
Secondly, did you checked your isolated RNA? You can easily do it by denaturing electrophoresis.
Third, why not adding RNase inhibitor since the beginning, in the RT buffer.
Last, in case of using either oligo dT or P6 primers, be careful of their concentration vs your RNA concentration. If too concentrated, P6 will direct revers-transcription of too short products that preclude further annealing of specific PCR primers.
Because your description is limited, if still unsuccessful, please come back with more details to allow us to figure out what was wrong with your reaction. Every step is important.