I work on microRNAs for my master thesis. Recently I've faced a problem in real-time PCR. I have 2 biological repeats and 3 technical repeats for each sample to detect the microRNAs expression level. but the biological repeats show almost meaningful difference with each other. I have collected my samples from the 4 upper leaves of the plant (pooled the 4 upper leaves of each pot as one sample). But I still doubt if this problem does originate from sampling or some where else. If you have had same experiences working with miRNAs, please let me know where the problem may lay.
Firstly, I think, You should do the experiment 1 more time..i.e the 3rd biological replicate, before trying to find out, would it because of sampling error or because of experimental error.
Secondly (This is optional) If possible include "spike-in" control (exogenous RNA) while processing your RNA isolation. Basically, you need to introduce a known concentration of RNA in every samples just before processing/or during processing. So, after Real time PCR, the amplification of spike-in RNA would produce more or less equal Ct value. It would provide you a good normalization control and also help you to know if there is any experimental error. Its been a recent trend now a days and there are many articles available about it's actual detail experimental applications.
Thank you so much Rakesh Dey, fortunately I have 2 other biological repeats in the -70 fridge, so I may use them to make sure about the sampling error. actually I've done the dilution as you mentioned before cDNA synthesis (200ng RNA) for all the samples. And yes, including a house keeping gene would be a great idea. I've already ordered my primers for 18srRNA. If you have any information about the normalization process in this method, I'd appreciate you to let me know.
Hi Shirin, what i tried to say was, to include an "spike-in" exogenous RNA control. So in these case, the primers for endogenous genes (your test genes) will not amplify or interfere the amplification of exogenous RNA and similarly, the primers for exogenous gene will not amplify any endogenous test genes (theoretically). So this way, it will serve as an additional normalization control besides any endogenous house-keeping controls (like 18S rRNA, GAPDH or beta-actin etc. etc.). Actually, this serve purpose, when endogenous controls also vary too much in sample to sample.
i hope, i could make it more clear now :-)
I think I got you this time :)
thank you. I'm gonna google to find more information in this regard.
Hi Shirin, I don’t think 18S-rRNA is a good choice for normalization of miRNA expression data and by using that I’m afraid you may get a false interpretation of your results. For normalization of miRNA expression values only miRNAs can be used because other types of RNA like mRNAs or ribosomal RNAs are different and therefore have different degradation capacity. For more information I recommend this link: http://www.biogazelle.com/how-find-stably-expressed-micrornas and also this paper: Meyer et al. (2012) Profound effect of profiling platform and normalization strategy on detection of differentially expressed microRNAs--a comparative study. PLoS One. 2012;7(6):e38946. doi: 10.1371/journal.pone.0038946.
Hi Arash,
Thank you so much for your response. I'm gonna read the paper and the link was quite a good clue.
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