I used verso cDNA synthesis kit, the RNa used had following concentrations
sample1: 485ng/microlitre with A260/280: 1.9.
sample2: 981 ng/microlitre with A260/280: 1.75
In order to check the primers of my gene for annealing temp i set up a simple gradient PCR, but was unable to obtain any bands on 2% gel.
What could be the possible reason for reaction failure?
Should i now go ahead for Real Time PCR?
- Please help.
Running RNA on gel you should see two well formed bands (the two subunits of ribosomal RNA). It is at least some index that your RNA is intact. And the rule is that the upper band should be twice more than the lower... take a look on the attached pic ...
What was your 260/230 values from your RNA isolation? A low 260/230 value indicates possible contaminants (phenol, salts, ethanol etc) which may be interfering with the RT reaction. RNA cleanup may be advised if this is the case.
I agree with Robert, you should try a primer pair that you know is working as a positive control. If you get a band there, you can rule out problems with the cDNA synthesis.
Also, if you establish new primers you can try different MgCl2 concentrations in addition to a temperature gradient (e.g. 1, 1.5, 2, 2.5 mM). You should, however, be sure that the gene you want to amplify is expressed in you RNA sample.
Only if you get one clear band in the regular PCR you should proceed with real-time PCR.
Good luck,
Britta
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