Two bands on RT-PCR product - (May/11/2006 )
Hi,all. I isolated RNA from using QIAGEN RNeasy kit and performed RT-PCR using QIAGEN onestep RT-PCR kit. The cycle I performed is 30 min at 50C for reverse transcription, 15 min at 95C for Initial PCR activation step, 3 step cycling which include 30 sec at 94C, 30 sec at 60C, 1 min at 72C for 40 cycle. And 10 min at 72C for final extension. I got two unexpected bands that are much higher than what it is expected. I should get the product about 440 bp, but I got two bands about double sizes. Could you suggest what those bands are and what did I do wrong. I really appreciate having your comments. Thanks.
are you amplifying gDNA? do your primers span introns?
No.. my primers doesn't have any intron and I amplified mRNA...
You should either have primers that span an intron or you should digest your RNA with DNAse (RNAse free) before RT-PCR.
In the next run you can include one sample with your primers and gDNA, just to see wether yoiu have DNA contamination in your RNA.
If you really want to know what it is: extract band from gel and send it to sequencing.
No.. my primers doesn't have any intron and I amplified mRNA...
You should either have primers that span an intron or you should digest your RNA with DNAse (RNAse free) before RT-PCR.
In the next run you can include one sample with your primers and gDNA, just to see wether yoiu have DNA contamination in your RNA.
If you really want to know what it is: extract band from gel and send it to sequencing.
Thanks for your suggestion. According to the RNeasy kit from QIAGEN, DNase I won't need actually. However, they add optional stage for treating DNase I, if we really concerned about RNA. I think I should use DNase during RNA isolation using this QIAGEN kit.
again, I think your most important thing is to have intron-spanning primers, if it's at all possible!
Hey,
Yep. Use the DNase step. It really helps. Run your extracted RNA on formaldehyde denaturing agarose gel to see if its really pure. you should see only the RNA bands and nothing else. I think it looks a bit like contaminating gDNA or other kinds of RNA. Perhaps you may try 2 step?
Regards,
Chris 
If you are using the RNeasy kit you should definitely carry out a DNase treatment. The DNA levels are less than or equal to 5% with silica methods, which is more than enough to amplify in PCR, and is probably equivalent to the amount of mRNA present in the total RNA sample
Thanks all.. I got lots of suggestions from you.
I will do RNA extraction again with DNase. Have a nice day.