I isolated RNA from pooled samples of mice retina (frozen samples). The RNA quality was fine. But, I am not able to detect the expression of a particular gene while the others were able to detect the expression. I am not able to speculate what can be the reason behind it, is it due to the quality of the RNA or the handling. Please help.
Hi Rishabh,
to really help you here I have some questions.
1. How have you analyzed the quality of your RNA? By photometry or did you also run the RNA on a gel. To be sure to have intact RNA you should not rely on only photometer data but also do gel and see if you can see the28s and 18s clealrly with only some smearing (the photometer can give good data regarding purity even when the RNA is relativly degraded). If you haven't done this please do so (maybe you than show the gel)
2. Do you use primers you have designed on your own or primers which were described elsewhere? Check the primer sequences even if there were described in another publication - sometimes there are mistakes.
3. Do you have a good working positive control for your assay? You should always include a positive control to see if your template integrity is good (you should also always include a negative/no template control to check for contamination)
Only when you have verified that you have a good template (gel + good working positive + negative control) you have to check your PCR.
- Make sure that all your reagents are working (see positive control)
- Make sure your primer are intact
- Work on your PCR protocol e.g. on the annealing temperature
I hope this helps and feel free to ask question - may be I or some other researcher can help.
Good Luck