Hello Researchers,
In most of the articles for RT-PCR of exosomes its suggested to take RNA input as volume (say 6 ul) with addition of spike-ins (for normalization of RT-PCR) and not amount due to the less concentration of exosomal RNA isolated from the biofluids. My query is that how will the variable amount (say 20ng or 200 ng) in that 4 ul of RNA input volume will be adjusted by spike-in? Does the same goes for supernatant derived exosomal RNA also?
Hi Nisha
If I understand your question correctly it won't do. The purpose of the spike in is for a quality control across your samples for the RTPCR reactions. it wont provide any normalisation to adjust for variation in RNA concentrations within your 6ul volume. The addition of spike ins will have the effect of lowering the concentration of the original RNA concentration in your samples. But that's ok. You can just normalise using a housekeeping gene.
Agree with Simon,
the correct internal control should demonstrate the changes to the same extent as the total amount of RNA in the sample. You cannot use the spike-in control because you manually add the same amount of this RNA to different samples. At the same time, there are no standard housekeeping RNAs in the exosomes. Working with exosomal RNA should always begin with the selection of the most suitable normalization method for the given samples.
You can check this paper as an example:
Faraldi, M., Gomarasca, M., Sansoni, V. et al. Normalization strategies differently affect circulating miRNA profile associated with the training status. Sci Rep 9, 1584 (2019). https://doi.org/10.1038/s41598-019-38505-x
Thank you, Simon Wyn Jones and Vasily A Yakovlev for your reply. I agree with your reply. Sir, but it is rather difficult to find an endogenous exosomal miRNA for normalization based on literature also as the sample collection, types of sample, etc. vary from that mentioned in the literature. Also, it's not possible to verify 5-7 endogenous miRNA for normalization on the sample. Then how have so many publications been accepted with the spike-in for normalization?
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