real time RT-PCR in bacteria - quantitative PCR (Aug/24/2005 )
I am trying to analyze mRNA levels of two proteins (auracyanin A and auracyanin B) to see relative expressions from the Chloroflexus bacteria.
I am using these on my two-step realtime RTPCR protocol:
BioRad iScript for reverse transcription
BioRad SyBr Green supermix for real time PCR
BioRad MyIQ Machine
I designed primers for about 170 bp region of the following:
gene1: auracyanin A
gene2: auracyanin B
endogenous control: 16s rRNA gene
Problems I encountered:
1. On the realtime PCR, I get signals from a well where I add RNA as template (as control). Also, I get signal from the no-reverse-transcriptase-control of the RT reaction. Do I have to use an additional RNase step to get rid of RNA after reverse transcription before I go to the real time PCR?
2. I get dimers in 16s rRNA gene. Is there a way around this without having to redesign the primers? Or is it even worth it? I only want to measure the relative expression of the two genes on cells grown in different conditions. Should I just use the Pfaffl or delta delta Ct method (but I am not sure of my efficiencies yet).
3. I have not encountered many quantitative PCR papers on bacteria so I just adapted the 16s rRNA gene from the 18 srRNA gene of eukaryotes. Does anyone work on bacteria as well, and what standards are accepted?
I would really appreciate any help. Thanks.
I am going to start RT-PCR and I do work with bacteria too. May it be that your samples or chemicals are contaminated?
As for the standard: I will use in vitro transcriped RNA from my target gene. This synthetic RNA carries the same primer binding sites as my target gene and thus, hopefully, will ensure an equal amplification efficiency of the target and standard.
There are a few very good papers out there about RT-PCR on bacteria. Search for Sharkley et al. (2004) "Detection and Quantification of Gene Expression in Environmental Bacteriology" - just have a look at the references and you will find a couple more interesting papers. Also, check this one out: Fey et al. (2004); key words Real-time PCR & Salmonella.
Hope that helps a bit.
thanks for the help! after asking some peers, i am convinced i have dna contamination on my rna samples and should treat it with dnase prior to reverse transcription. i also am re-designing my primers so they won't dimerize. thank you for the references, i am printing them now.