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Comparison and optimisation of microRNA extraction from the plasma of healthy pregnant women.

Author(s): Parker VL, Cushen BF, Gavriil E, Marshall B, Waite S, Pacey A, Heath PR

Publication: Mol Med Rep, 2021, Vol. 23, Page

PubMed ID: 33576446 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the microRNA (miRNA, miR) yield and levels of individual miRNAs after isolation using the Maxwell RSC and miRNeasy Kit with and without inclusion of Proteinase K or RNA carriers (MS2 or glycogen) from 100-500 µL plasma of a pregnant woman. Blood was collected using a 21-gauge needle into sodium citrate vacutainer tubes from a pregnant woman during the 9’th week of gestation and stored upright on ice for <2 h. Plasma was obtained by centrifugation at 1,900 x g for 10 min at 4˚C and stored frozen at -80°C. Specimens were thawed at room temperature and platelet-poor plasma (PPP) was obtained by centrifugation at 16,000 x g for 10 min at 4˚C. RNA was extracted from PPP spiked with UniSp2, UniSp4, and UniSp5 spike‑in mix using the miRNeasy Serum/Plasma Kit with different plasma volumes (100, 200, or 500 µL), with or without 60 µL Proteinase K or the RNA carriers MS2 (2.5 µg) or glycogen (5 µg) and with the Maxwell RSC miRNA from Tissue and Plasma or Serum Kit using 100 or 200 µL plasma with or without addition of 24 µL Proteinase K, MS2 (0.5 or 1 µg), or glycogen (2 µg). The percentage of small RNA that was miRNA and miRNA concentration were assayed using an Agilent Small RNA Chip on a 2100 Bioanalyzer. Levels of hsa‑miR‑222‑3p, hsa‑let‑7i‑3p, hsa‑miR‑148‑3p, and hsa‑miR‑30e‑5p were quantified by real-time RT-PCR. Additionally, extraction efficiency was quantified by real-time RT-PCR amplification of UniSp2, UniSp4, and UniSp5.

   Summary of Findings:

   While variable among the optimized methods, miRNA concentration and percentage were comparable between the two extraction kits. However, recovery of the spiked-in controls was less consistent using the Maxwell RSC than the miRNeasy Kit with a fold difference in recovery of spiked-in controls of 5.63- 8.05 and 6.69-7.88, respectively, and a variance in Cqs of 2.96-5.26 and 0.72 -1.90 cycles, respectively. Welch’s ANOVA analysis showed a significant effect of plasma input volume on normalized levels of hsa‑miR‑222‑3p (P=0.02) and inclusion of Proteinase K, MS2, or glycogen on normalized levels of hsa‑miR‑148‑3p (Welch's ANOVA, P=0.0006) and hsa‑miR‑222‑3p (Welch’s ANOVA P=0.046). Pairwise analysis found higher normalized levels of miR-222-3p with an input volume of 200 µL instead of 100 µL using the Maxwell Kit with no proteinase or carriers (P=0.02) but no differences were found using the miRNeasy Kit. Higher normalized levels of miR-222-3p were found when extraction was from 200 µL of plasma with miRNeasy (no carrier) than when from 100 µL plasma using the Maxwell Kit without a carrier (P=0.02). In pair-wise comparisons, inclusion of Proteinase K along with glycogen or MS2 in the Maxwell Kit led to higher normalized levels of hsa‑miR‑148‑3p than when extraction included MS2 but not Proteinase K (P=0.04 and P=0.048, respectively), but no significant differences in normalized hsa‑miR‑148‑3p levels were found with inclusion of Proteinase K, MS2, or glycogen when extraction was with miRNeasy. Pair-wise comparisons did not identify any significant differences in hsa‑miR‑148‑3p with inclusion of Proteinase K, MS2, or glycogen. Importantly, while there was no difference in normalized levels of the four miRNAs between kits when extraction was performed using the manufacturer specified conditions, use of the Maxwell Kit rather than the miRNeasy Kit resulted in significantly higher variance in normalized levels of let‑7i‑3p (P=0.03), hsa‑miR‑30e‑5p (P<0.0001), and miR‑222‑3p (P=0.003). UniSp6 Cq values were higher in the no template control than the samples but a comparison of the global mean UniSp6 values versus inhibition controls did not find a significant difference. Further, no hemolysis was found.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Pregnant

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Aliquot size/volume	100 µL 200 µL 500 µL
   Real-time qRT-PCR Specific	Targeted nucleic acid	hsa‑miR‑222‑3p hsa‑let‑7i‑3p hsa‑miR‑148‑3p hsa‑miR‑30e‑5p
   Analyte Extraction and Purification	Protein digestion	Proteinase K used No proteinase K used
   Analyte Extraction and Purification	Analyte isolation method	miRNeasy Serum/Plasma Kit Maxwell RSC miRNA Tissue or Plasma or Serum Glycogen added MS2 added No Glycogen or MS2 added

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