RT-PCR: Band in no reverse transcriptase lane - (May/07/2007 )
Any ideas on what's going on? I'm trying to do RT-PCR for Myc2. I keep getting a band for my negative control (no reverse transcriptase). However, this doesn't happen with the actin RT-PCR using the same reverse transcriptase reaction.
write your problem in detail. there might be any contamination in your negative control, as u said that there is no band in Actin it means there might be contamination in your primer. try to use other aliqoute and see.
all the best
Sorry, here's what's going on:
I'm developing an RT-PCR assay for Myc2. My reverse transcription reaction consists of RNA (from mouse lymphocytes), RNASin, random hexamers, RT buffer, and RT. My negative control is an identical reaction lacking RT.
My PCR for Myc2 results in a band from the no-RT negative control, equal in size/intensity to the band from the experimental reaction. I've tried this with three different primer sets, with the same result.
When I PCR for actin, there is no band from the no-RT control.
Any troubleshooting ideas would be greatly appreciated. Thanks!
Reverse transcriptase is really sensitive. I am suspecting your initial source instead. 
Have you checked if your primers are specific for mRNA and not match genomic DNA?
Maybe you could have a genomic contamination in your RNA extraction, and so you see a band in NO RT sample.
I had the same problem, but my primers checked also genomic DNA...
try to perform DNAse digestion after your extraction and see if you'll have the same problem.
Thanks for your help, Sandy. My primers should be specific for mRNA. I'm also suspecting some DNA contamination. I use TRIzol for my RNA extraction, if that helps.
I'm trying another set of primers which should be more fussy over cDNA vs. gDNA, plus I'll try DNAse treatment.
I'm trying another set of primers which should be more fussy over cDNA vs. gDNA, plus I'll try DNAse treatment.
This strategy sounds good. Start with the optimization of RNA extraction and purification (do you have good spec readings?) + DNAse treatment before ordering new primers! To rule out primer contamination, use No Template Controls. Are your primers spanning exons?
I'm trying another set of primers which should be more fussy over cDNA vs. gDNA, plus I'll try DNAse treatment.
This strategy sounds good. Start with the optimization of RNA extraction and purification (do you have good spec readings?) + DNAse treatment before ordering new primers! To rule out primer contamination, use No Template Controls. Are your primers spanning exons?
My 260/280 ratios are in the 1.8-2.0 range, so I think my RNA is good. One primer straddles an exon/intron junction, the reverse primer is well into the following exon--this gives me a much larger product than I generally see used in the literature but I'm thinking it will be more cDNA specific.
Thanks for your help!
Not at all! Afaik, pure RNA should have a value of 2.1 (pure DNA of 2.0), RNA extraction with Trizol usually has a value of 2.0-2.1 by optimal starting material. I always include a DNase step, but just test a DNA sample, if you see 'your' band again, it would be the easiest way to get rid of it.
1.8-2.0 is the middle range. Above 2.0 will be good for RNA. Below 1.8 will be protein contamination. Isnt it?
Yea, agreed with Horlem, always use Dnases to check your RNA quality. Again, during this step, better be careful not to introduce any trace of RNases. 