I'm currently working with an organism which has no established endogenous control mirna that I could use to normalize qpcr data. I'm thinking about instead using an exogeous control mirna such as the ce-39 mirna to normalize quantitative data. I'm thinking it would be best for my purposes to spike it in before rt/cdna synthesis but I also see many papers where it's spiked in during the rna isolation step.
So I suppose I have two questions:
1) Is it valid for me to use an exogenous control to normalize qpcr data when I have no real other option?
2) Can I simply put it in before the cdna synthesis step and then compare the mirna results of my gene of interest to it? Or do I need to put it in during the rna isolation step?
I have seen it mostly as an rna isolation spike-in but this was also when they couldn't control for the amount of rna being put into the cdna synthesis, whereas I am able to obtain enough rna (I'm not using plasma/serum, which is known to be difficult to obtain mirna from) to control for the amount being put into cdna synthesis.
Thanks!
This will be relative. From everything I've seen, even relative quantification requires something stable to compare it to (ie an endogenous control in most species where we know the endogenous control genes). However in my species we don't really know or have the information to know what good control genes would be. Instead I've been seeing people spike-in a mirna from a separate speces (ie the ce-39 mirna) which should be good to compare it to. I suppose my next question would be, is this ok?
Normally, you would culture the bacterial strain, extract genomic DNA and create a standard curve. Do you have any possibility to culture ?
If you wanted to generate a standard without the cultivation I would recommend you to run a PCR of a positive sample using specially designed primers. I would then amplify the bacteria of the former PCR using the same primers in a comparable PCR setting. Primers and PCR settings can be found throughout the scientific literature I suppose. Then I would quantify the amount of DNA using e.g. photometer/fluorometer. With this you might be able to generate standard curves (see here for example: http://www6.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf).
If you are ok to do relative quantification (x-fold difference between samples) there are lots of protocols regarding the delta delta CT method that do not require a standard curve.
Hope this helps a bit. If not feel free to contact me any time.
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