I tried isolating total RNA from serum and plasma using Qiagen miRNeasy kit and estimated its concentration. The serum concentration was 0.15ng/ul and plasma concentration was 2.07ng/ul. We are trying to find which one (serum/plasma) is most suitable for downstream applications. I tried performing qPCR using an endogenous control (mir-423-5p) for both serum and plasma samples using Taqman miRNA assay. The protocol states using 2ul of starting sample, but is it required to take an equal concentration of both? I just went along with the protocol and got Ct values for both serum and plasma performed as 4 replicates. I now want to establish this for target genes suitable to study serum and plasma. What miRNA target genes can be used to study the relative expression? Inputs and suggestions are appreciated!
Select the gene list tab from the link below.
https://www.qiagen.com/au/shop/pcr/primer-sets/miscript-mirna-pcr-arrays/?catno=MIHS-106Z#geneglobe
"Routinely Detectable in Normal Serum: miR-103a-3p, miR-15b-5p, miR-16-5p, miR-191-5p, miR-22-3p, miR-24-3p, miR-26a-5p."
Always stick to the manufacturers protocol. I would take 4-5 miRs from that list and run a reference gene stability check using a software (i.e. geNORM, normfinder). Some Bio-Rad CFX models have a built-in software for this. I would also read the article below
http://www.rdml.org/clinchem.2008.112797v1.pdf
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