Real Time PCR : Bacterial gene quantification, can it be done? - (Nov/20/2006 )
I am using real time pcr to investigate the regulation of a gene in a lactic acid bacteria.
I have isolated RNA (Trizol, Invitrogen) and DNAse treated it (DNA-free, AMBION).
I initially reverse transcribed RNA using a Taqman kit and then used SYBR Green Master mix for the PCR step. I observed variation of the CT value within replicates, my NTC was postitive towards the end of the run (i know this is acceptable when >30) and my samples of interest and No-RT control had the same CT value.
I changed to the Taqman probe approach, this lead to a negative NTC, my CT values were identical within replicates and between experiments. However my No-RT control is still postivive. ABI told me to use a cDNA specific primer and probe. I cant do this with bacteria as they are prokaryotesnd do not have introns (The cDNA specific approach only works for eukaryotes as the primer and probe are at the end of one exon and the next primer is one the next exon, when the intron is removed during post-transcriptional modification the primer and probe will be able to amplify only cDNA as the genomic DNA has the intron interupting the exons).
I know my DNAse work as it gives a shift to the right with a delta CT of 8.6 (~388 fold reduction) which is fine. But i still see genomic DNA contamination. I could try a second DNAse treatment but to my mind if real time is powerful enough to detect a single copy of DNA i will alway achieve a postive result in my No-RT contol.
Because cDNA and genomic DNA are the same the primers and probe will amplify from both and give the same signal, therefore i cannot be sure that i am seeing a change in gene expression or just a change in the level of contamination between samples.
I have checked the literature and have found a few papers (3) on real time PCR and bacterial gene quantification in terms of expression. The majority of papers look at population quantification or the effect a bacteria has on the gene expression in a eukaryotic model.
So i have the following questions:
1. Is it possible to quantify bacterial gene expression using Real Time PCR, even though RNA is contaminated with genomic DNA and no DNAse is 100% efficient?
2. Has anyone else tried to quantify bacterial gene expression and willing to tell me about their No-RT controls?
3. Any suggestions on reducing DNA contamination?
You have to improve the RNA purification set. We use a kit from invitrogen that is an on column rna purification with a dnase treatment. after that we do another dnase treatment. this normally gives a very pure RNA.
About the No RT controls. mine are usually negative (no detection). Althought i´ve had positive readings when the anealing temperature was not the best one. I would suggest that you try your Rt-pcr primers on genomic DNA and do a gradient PCR to determine the best anealing temp, use this temp on the RT-PCR cycle and see if it improves the no-Rt control.
Also check that your primers don´t form dimmers, which would also explain the positive readings on the no-RT (have a look at the melting curve- this is an option we have when doing RT qPCR using the Opticon system, don´t know if its the same for everyone...)
Hope this helps!
Dosen't bacterial RNA degrade quickly due to the lack of a polyA tail? In this case I would add something like RNAlater to the sample before.