Comparison of miRNA quantitation by Nanostring in serum and plasma samples.
Author(s): Foye C, Yan IK, David W, Shukla N, Habboush Y, Chase L, Ryland K, Kesari V, Patel T
Publication: PLoS One, 2017, Vol. 12, Page e0189165
PubMed ID: 29211799 PubMed Review Paper? No
Purpose of Paper
This paper compared the microRNA (miRNA, miR) profiles of matched serum and plasma and investigated the effects of different normalization strategies.
Conclusion of Paper
Of the 427 miRNAs detected, 266 were found only in plasma, 10 only in serum, and 151 in both serum and plasma. The number of miRNAs with counts >5 was variable between patients but was higher for plasma than serum; however, the average miRNA count was slightly higher in serum than in plasma. Serum had low but consistent expression of all five of the endogenous control mRNAs, but three of the five were more abundant in plasma although more variable between patients. Using geNorm, miR-95-3p was identified as the most stable miRNA, but the average count in serum and plasma was less than 5. The list of the 10 most stable miRNAs with counts >5 differed between serum and plasma, but miR-30e-5p was found to be the least variable in both. For serum, the lowest CV for serum was found when normalized to the median rather than miR-95-3p, miR-30-5p, or the three most stable miRNAs in serum. In contrast, the lowest CV for plasma was achieved through normalization to miR95-3p or the three most stable miRNA. Levels of the white blood cell (WBC)-specific miRNA, miR-24-5p, were elevated in 7 of 28 plasma specimens but only 2 of 28 serum specimens, indicating potential WBC contamination of the plasma. The authors report the number of detectable miRNA species was not correlated with patient gender or RNA concentration.
Studies
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Study Purpose
This study compared the microRNA (miRNA, miR) profiles of matched serum and plasma specimens and investigated the effects of different normalization strategies. Blood was collected using a 21-gauge needle from 3 patients with benign disease (non-alcoholic steatohepatitis or primary sclerosing cholangitis) and 25 patients with primary liver cancer into Vacutainer serum and plasma tubes. Plasma was obtained by centrifugation at 2900 x g for 10 minutes at 4˚C within 2 h of collection, aliquoted, and frozen at -80˚C. Blood in serum tubes was allowed to clot for 30 min before centrifugation at 3000 rpm for 10 minutes at 4˚C and subsequent storage of the serum at -80˚C. After thawing, specimens were centrifuged at 3000 rpm for 5 min to remove residual cells. RNA was isolated using the miRCURY RNA Isolation Kit with the on-column DNase treatment. RNA was concentrated using the RNA Clean and Concentrator-5 Kit. RNA was analyzed by NanoDrop and expression was determined using the nCounter Human v2 miRNA Panel on a NanoString nCounter Analysis System.
Summary of Findings:
A total of 427 miRNAs were detected in serum and/or plasma. Of these, 266 were found only in plasma, 10 only in serum, and 151 in both serum and plasma. The number of miRNAs with counts >5 was patient-dependent and was higher for plasma (25-221 miRNA, median =91) than serum (3-82 miRNA, median = 17), but the average count in serum was higher than in plasma (19.44 versus 19.14). The authors report the number of detectable miRNA species was not correlated with patient gender or RNA concentration.
Serum had low but consistent expression of all five of the endogenous control mRNAs. In contrast, while plasma levels of RPL10 and GAPDH were very low and consistent between specimens, plasma levels of B2M, ACTB, and RPL19 were high but displayed more variability, especially in the case of B2M. Interestingly, three of the five non-endogenous miRNAs were found at much higher levels in plasma than serum but also displayed higher variability in these specimens. Using geNorm, miR-95-3p was identified as the most stable miRNA but the average count in serum and plasma was less than 5. The list of the 10 most stable miRNAs with counts >5 differed between serum and plasma, but miR-30e-5p was found to be the least variable in both. For serum, the lowest CV was found when normalized to the median rather than miR-95-3p, miR-30-5p, or the three most stable miRNAs in serum. In contrast, the lowest CV for plasma was achieved through normalization to miR95-3p or the three most stable miRNAs.
Levels of the WBC-specific miRNA, miR-24-5p, were elevated in in 7 of 28 plasma specimens but only 2 of 28 serum specimens, indicating potential WBC contamination of the plasma. Levels of miR23a-3p were not consistent so it was not possible to use the ratio of miR451a to 23a-3p to examine hemolysis.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Other diagnoses
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA DNA microarray Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) DNA microarray Specific Data handling Normalized to the median
Normalized to miR-95-3p
Normalized to miR-30-5p
Normalized to the three most stable miRNA in serum
Normalized to the three most stable miRNA in plasma
Biospecimen Aliquots and Components Blood and blood products Plasma
Serum