Assessment of six commercial plasma small RNA isolation kits using qRT-PCR and electrophoretic separation: higher recovery of microRNA following ultracentrifugation
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- Assessment of six commercial plasma small RNA isolation kits using qRT-PCR and electrophoretic separation: higher recovery of microRNA following ultracentrifugation
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Growing interest in blood-borne microRNAs (miRNAs) as biomarkers has led to the introduction of a number of commercial
kits for isolating small RNAs from plasma/serum. We sought to compare the efficacy of six such kits in isolating miRNAs
from either whole plasma or a plasma-derived ultracentrifugation (UC) fraction from 2 healthy volunteers with some of the
results being validated in 10 additional subjects. To assess the overall yield and concentration of isolated small RNAs, we
measured the levels of one spiked-in and four endogenous miRNAs by quantitative reverse transcription and polymerase
chain reaction (qRT-PCR). We also tested the performance of the Agilent Bioanalyzer small RNA assay with these RNA samples.
Additionally, we tested the effects of hemolysis on measured miRNA levels in whole plasma and in the UC fraction.
Both the efficiency of RNA isolation and the relative levels of specific miRNAs in different samples varied considerably between
the tested extraction methods. Of all kits tested, the QIAGEN miRNeasy kits (Mini and Serum/Plasma kits) and the
Macherey-Nagel NucleoSpin kit produced the highest RNA yields. The QIAGEN Exo kit produced lesser yields than what
could be extracted from the UC fraction using the QIAGEN miRNeasy kits and the Macherey-Nagel NucleoSpin kit.
Bioanalyzer results showed an average correlation of R2¼0.8 with endogenous miRNA qRT-PCR results, for sample concentrations
>40 pg/ml. The levels of the endogenous miRNAs measured in the two volunteer samples were compared with those
in a larger group of subjects (n¼10) and found to be typical. Our comparison favors the use of the QIAGEN Serum/Plasma kit
and the Macherey-Nagel NucleoSpin kit for plasma miRNA applications. Furthermore, extraction of miRNAs from the UC
fraction results in higher yield than extraction from whole plasma
Originalsprog | Engelsk |
---|---|
Artikelnummer | bpw003 |
Tidsskrift | Biology Methods and Protocols |
Vol/bind | 1 |
Udgave nummer | 1 |
Antal sider | 9 |
ISSN | 2396-8923 |
DOI | |
Status | Udgivet - 2016 |
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