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A comparison of the efficiency of RNA extraction from extracellular vesicles using the Qiagen RNeasy MinElute versus Enzymax LLC RNA Tini Spin columns and qPCR of miRNA.

Author(s): Dunlop RA, Banack SA, Cox PA

Publication: Biol Methods Protoc, 2021, Vol. 6, Page bpab015

PubMed ID: 34423131 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study used plasma collected from a single donor to compare methods of RNA extraction from EVs (Enzymax columns, RNeasy columns); Cq values of RNA controls spiked in prior to extraction (to assess extraction efficiency) or prior to cDNA synthesis (to assess the efficiency of cDNA synthesis) were compared as were Cq values of normal and low abundance endogenous miRNA. RNA was extracted using Enzymax columns orRNeasy columns. Effects of doubling the RNA input amount for the reverse transcription reaction on Cq values were also explored. Frozen plasma from a healthy donor was obtained from a commercial source. EVs were precipitated with SBI ExoQuick, collected by centrifugation at 1500 g for 20 min at 4°C. Neural EVs were removed by incubation with an antibody to L1CAM for an hour at 4°C, followed by incubation with streptavidin-agarose resin for 1 h; neural EVs were then pelleted by centrifugation at 200 g for 10 min. Non-neural EVs were spiked with miRCURY (UniSp2, UniSp4, and UniSp5) before RNA was extraction with the Qiagen RNeasy Midi Kit with the Qiagen miRNeasy spin columns or with the Enzymax RNA Tini Spin columns. The RNA extract was spiked with UniSp6 and cel-miR-39p and then reverse transcribed using the miRCURY LNA RT Kit. Levels of the spike-in controls, a panel of six miRNAs (miR-142-3p, miR-451a, miR-23a-3p, miR-30c-5p, miR-103a-3p, miR-191-5p), and ten medium to low abundance miRNAs (miR-146a-5p, miR-199a-5p, miR-146a, miR-4454, miR-10b-5p, miR-29b-3p, miR-151a-3p, miR-199a-3p, miR-155a-5p, miR-126-5p) were quantified by real-time PCR using miRCURY LNA miRNA SYBR PCR Assays.

   Summary of Findings:

   As expected, adding twice as much RNA input to the reverse transcription reaction increased the levels of the RNA spike-ins (0.83-1.28 Cq) as well as levels of the six targeted miRNAs (1.10-1.71 Cq); however, Cq values of the RNA spiked immediately prior to reverse transcription reaction was not altered by input amount (-0.33 to 0.21 Cq). Cq values for the RNA extraction control spike-ins and the six targeted RNA were lower (𝛥Cq of ≤-1.05) when the RNeasy columns were used rather than Enzymax columns, but the differences were variable and not statistically significant. Importantly, no significant effect of spin column type was found on the Cq values of the ten medium to low abundance miRNAs.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Qiagen miRNeasy spin columns Enzymax RNA Tini Spin columns
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-142-3p miR-451a miR-23a-3p miR-30c-5p miR-103a-3p miR-191-5p miR-146a-5p miR-199a-5p miR-146a miR-4454 miR-10b-5p miR-29b-3p miR-151a-3p miR-199a-3p miR-155a-5p miR-126-5p
   Real-time qRT-PCR Specific	Template/input amount	2 µL RNA 4 µL RNA

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