I'm intending to perform a ddPCR to check miRNA expression patterns in human blood plasma. However, I am not sure which housekeeping gene I should use. The U6 is very differentially expressed in human blood plasma. Moreover in literature also other microRNAs have been proposed to be used as a housekeeping gene (miR-16, global normalization, miR-191-5p, miR-93-5p, miR-423-3p etc.), but none are conclusive. So is anyone else trying to measure microRNAs in human blood plasma via ddPCR and has a tip/idea which microRNA to use as a housekeeping gene?
I am also wondering whether a housekeeping gene is even needed when using droplet digital PCR? (according to Ma et al. in Biomarker Insights 2013, ddPCR does not require endogenous controls).
And if it is not needed is using a spike-in (miR-39 c.elegans) advisable?
Hi Charlotte
Despite of the method that you use for quantification, you need to consider that a housekeeping signal may have relative constant expression levels across samples. In ddPCR the requisites of normalizers are the same than for other quantitative PCR-based techniques. U6 is not a good housekeeping for plasma RNAs, since this RNA is intracellular, and its secretion is accidental. About the other normalizers already described in literature miR-16 is the most widely used, however in my hands has also problems depending on the cell type that you are analyzing. You can analyze your cells and determine which miRNA can be used as normalizer, however this protocol will involve screenings and is tedious and expensive.
I would recommend you to use a spike-in approach, seeding serum samples with a totally unrelated miRNA as you pointed out. This spike-in RNA can be used as a normalizer, but also as a control for RNA extraction and cDNA synthesis. In my hands works pretty well...
Good luck.
Best
Paco
Hi again Charlotte
I forgot to answer to the last question. In fact, and theoretically, ddPCR do not require the use of internal normalizers for quantification. However, remember that in the case of plasma miRNAs you are not using a raw sample, but a pre-extracted one. In this case, I would advice to use at least something to control the quality of RNA extraction and cDNA synthesis, since plasma/serum are rich in RT inhibitors.
Best.
Paco
Dear Paco and Anja, thank you for your advise. It is highly appreciated!
I think I am going to use the spike-in method and measure the RNA concentration and purity via bioanalyser (up till now I measured this with Nanodrop, but I believe Bioanalyser is more precise) so I can try to make the input amount of the small RNA as equal as possible (measuring a few normalization genes is unfortunately not possible due to time and money constraints, otherwise that would have been nice)
The best to you too,
Charlotte
If you have the qPCR data ( CT value) try some algorithm that define the most stable miRNA among many. two algorithm is Normfinder and Genenorm
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA
- microRNA