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Improved protocol for plasma microRNA extraction and comparison of commercial kits.

Author(s): Sriram H, Khanka T, Kedia S, Tyagi P, Ghogale S, Deshpande N, Chatterjee G, Rajpal S, Patkar NV, Subramanian PG, Gujral S, Hasan S, Tembhare PR

Publication: Biochem Med (Zagreb), 2021, Vol. 31, Page 030705

PubMed ID: 34658646 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the percentage of miRNA relative to small RNA, miRNA yield, and the recovery of four endogenous miRNA among those extracted from plasma using four different kits (miRNeasy Serum/Plasma, miRNeasy Mini Kit, RNA-isolation, and Absolutely-RNA MicroRNA Kit from Agilent technologies). Effects on percent miRNA and miRNA yield as a result of increasing the ratio of denaturing buffer, washing the column twice versus once and eluting twice versus once were also investigated following extraction with the miRNeasy Serum/Plasma Kit. Whole blood from 30 healthy donors was collected into K₂EDTA vacutainer tubes. EDTA plasma was separated by centrifugation at 1,500 x g at 4°C. Plasma was stored at -80°C for 4-6 months. Plasma was thawed on ice and centrifuged at 3000 x g for 5 min at 4°C. Plasma was spiked with cel-miR-39-3p and miRNA was extracted using the miRNeasy Serum/Plasma kit with the MinElute Cleanup Kit, miRNeasy Mini Kit also with the MinElute Cleanup Kit, Agilent RNA-isolation Kit, and the Agilent Absolutely RNA MicroRNA Kit.  The extraction protocol using the miRNeasy Serum/Plasma was optimized using the plasma of 20 healthy volunteers by testing different ratios of denaturing buffer to plasma (the recommended 5:1 versus 7:1), washing the column twice rather than once, and elution twice rather than once.  RNA was quantified using the Agilent Small RNA Kit. Extraction efficiency of a spiked-in miRNA (cel-miR-39-3p) and endogenous miRNAs (miR-24-3p, miR-191-5p, miR-423-5p and miR-484) were evaluated by real-time PCR amplification. CT values of miR-24-3p, miR-191-5p, miR-423-5p and miR-484 were normalized to cel-miR-39-3p.

   Summary of Findings:

   miRNA extraction with the miRNeasy Serum/Plasma Kit resulted in the highest percentage of miRNA relative to small RNA compared to the other kits evaluated (P<0.005 versus all other kits). Percentage miRNA was comparable when extraction was with miRNeasy Mini Kit or the Absolutely RNA Micro-RNA Kit. While extraction with the miRNAeasy Serum/Plasma kit also generated the highest miRNA yield, differences were only significant when compared to the miRNeasy mini kit or the RNA isolation kit (P<0.001, both). Finally, cel-miR-39 normalized CT values for all four endogenous miRNAs were lowest when extraction was with miRNeasy Serum/Plasma Kit, but for each of the four miRNAs differences were only significant when compared with the Agilent RNA-isolation Kit. Compared to the original protocol (one wash, one elution), the percentage of miRNA and total miRNA yield after extraction with miRNeasy Serum/Plasma Kit increased when eluted twice (P=0.007 and P<0.001, respectively), but decreased when the column was washed twice (P<0.001 and P=0.003, respectively). Non-significant declines in the percentage miRNA and miRNA yield were observed when the ratio of denaturing buffer used in the miRNeasy Serum/Plasma kit was increased.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	miRNeasy Serum/Plasma kit miRNeasy Mini Kit Agilent RNA-isolation kit Agilent Absolutely-RNA MicroRNA Kit miRNeasy Serum/Plasma kit with standard protocol miRNeasy Serum/Plasma kit with 7:1 denaturation buffer miRNeasy Serum/Plasma kit with two column washes miRNeasy Serum/Plasma kit with two elution steps
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-24-3p miR-191-5p miR-423-5p miR-484 cel-miR-39-3p

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