I have no expertise with PCR at all, but I do understand the principle behind PCR, qPCR and RT-PCR. I want to estimate the expression of certain gene in bacteria. But right now, I only need a rudimentary estimation. If the results are encouraging I can buy the kits required for qRT-PCR. I have two questions:
1. Can I conduct RT-PCR (Reverse Transcriptase) method in a regular thermal cycler? My limited knowledge tells me that it should be possible and I only need to add Reverse transcriptase to the master mix to get the cDNA from mRNA (and add a phase for reverse transcription to occur).
2. Will it be possible to make quantification of the cDNA level of the final PCR product to estimate gene expression level? My understanding is that qPCR determines the amount of cDNA at the end of each cycle. I don't need that. I just need to know the amount at the end of PCR and compare it to a housekeeper to make a relative calculation, right? Say, I want to measure expression levels of NorA, I will evaluate the amount of NorA cDNA at the end of the PCR reaction and compare it with amount of cDNA of rpsL (housekeeping gene). That should give me a ratio. Then I will measure the ratio for control bacteria as well and compare the ratio from sample bacteria to control bacteria to make a rudimentary guess about whether overexpression has occurred.
Does this make sense? [Sorry for the long post]. Again I don't know all the intricacies of PCR, so any comment on this would be highly appreciated.
With a Reverse Transcription PCR, you can semi-quantitatively understand the gene expression. You also need to perform a PCR for a house keeping gene, such as GAPDH, than after normalizing you can estimate the expression of your gene of interest.
In qRT( Quantitatively Reverse Transcription PCR) you use basicly a dye like SYBR Green or a DNA prob( FAM flourecent) than after each cyle system measure the relative flourecent amount. Ones the flourecent amount is doubled, PCR system detect it and you get expression datas of your gene of interest quantitatively.
In basic, RT pcr you compare the bands via your eyes or the programs that work as your eyes. But in qRT machine calculate amount of product via flourecent unit.
In short, NO.
You will see old publications that use so-called semi-quantititive PCR to measure gene expression. Basically, they make cDNA from the mRNA and stop the PCR machine at different cycles and look at band intensity to estimate gene expression. We know know that that method is inexact and honestly no better than a guess.
If you want to get anything published, you need to use real-time qPCR and/or RNAseq.
Your other choice would be to use agarose gel electrophoresis, nowadays this method is antiquated by the advent of RT-qPCR as mentioned above.
How to do this - run PCR, make gel, run the gel with appropriate ladders, image analysis on gel bands (like in Western blot).
Thank you all for your inputs. I want to stress that I am not thinking about publication right now. I just need a sign that I am going in the right direction. A rudimentary indication of gene over-expression will suffice for now.
If I get encouraging results, I will collect necessary kits for qRT-PCR.
Then go for the above-mentioned semi-quantitative RT-PCR. You can perform the RT in a standard thermal cycler (even a water / dry bath in extreme cases) because it only needs a step at 42°C and one at 70°C. Then you can do a standard PCR in a quite big volume (50 -100 µl) for your gene of interest and a housekeeping gene and take a small reaction volume (2-5 µl) of your samples every 5 cycles after 15 cycles and load them on a agarose electrophoresis gel. If you have a good gel analyzer, you can have an idea of your gene over-expression by looking for the ratio between bands intensities. Good luck!
Dear Chatonnet,
Thank you very much. Also thanks to others for their input..
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