Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Variability in microRNA recovery from plasma: Comparison of five commercial kits.

Author(s): Brunet-Vega A, Pericay C, Quílez ME, Ramírez-Lázaro MJ, Calvet X, Lario S

Publication: Anal Biochem, 2015, Vol. 488, Page 28-35

PubMed ID: 26271186 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared extraction methods on the quantification of endogenous and spiked-in miRNA in plasma from patients with colorectal cancer and healthy individuals. Peripheral blood was collected into EDTA vacutainer tubes from four patients with colorectal cancer and four healthy patients. Plasma was obtained by centrifugation at 3500 x g for 10 min at 4˚C, aliquoted, and stored at -70˚C. RNA was extracted by six methods including the miRCURY RNA isolation kit, Plasma/Serum Circulating and Exosomal RNA Purification Mini Kit, NucleoSpin miRNA Plasma Kit, miRNeasy Serum/Plasma Kit, and Direct-zol RNA MiniPrep. Synthetic UniSp2, UniSp4, and UniSp5 were added to the tube immediately after addition of the first buffer in the extraction method. Eluted RNA was aliquoted and stored at -70˚C. RNA was spiked with cel-39-3p and UniSp6 and was reverse transcribed using the miRCURY LNA Universal RT microRNA PCR Kit and used immediately for real-time PCR individual assays and specimens from six additional patients were also analyzed using the miRCURY LNA microRNA QC PCR Panel.

   Summary of Findings:

   The lowest cycle threshold (CT) values for the RNA spiked into plasma were obtained when extraction was with the miRCURY kit, but even the lowest abundance spike in miRNA was detected in all specimens. CT values for UniSp2, UniSp4, and UniSp5 were significantly lower using the miRCURY kit than the NucleoSpin Kit (P<0.05, all) and UniSp5 CT values were significantly lower using miRCURY kit than the Circulating and Exosomal RNA purification mini kit (P<0.05). CT values were strongly to very strongly correlated among the spiked RNA species (r=0.770-0.931, P<0.01). Based on the spike-ins added to extracted RNA, the reverse transcription reaction and PCR occurred with similar efficiency in all specimens, regardless of extraction method. Hemolysis, as determined by the difference in CT of miR-23a and miR-451, was slightly higher in specimens extracted using the Circulating and Exosomal RNA kit, but the differences were not significant. While levels of miR-103, miR-191, and miR-30a were unaffected by extraction method; levels of miR-23a were strongly correlated with extraction method used (r=0.879-0.957, P<0.01) and levels of the hemolysis marker, miR-451, were modestly correlated with extraction method (r=0.433-0.516, P<0.05). The within-specimen variability in levels of the spiked-in RNA was high and levels of RNA were higher in specimens from patients with colorectal cancer than from healthy controls. Levels of miR-29a and miR-103 were unaffected by extraction kit used, but miR-21 mean CT values were higher when miRNA was extracted using the NucleoSpin miRNA kit rather than the miRNeasy or Direct-zol kits (P<0.5), and miR-18a and let-7a mean CT values also depended on the extraction kit used.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   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	miRCURY RNA Isolation kit Plasma/Serum Circulating and Exosomal RNA kit NucleoSpin miRNA Plasma kit miRNeasy Serum/Plasma Kit Direct-zol RNA MiniPrep kit
   Real-time qRT-PCR Specific	Targeted nucleic acid	UniSp2 UniSp4 UniSp5 UniSp6 cel-39-3p miR-23a miR-29a miR-451 miR-103 miR-191 miR-30a miR-18a let-7a

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