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Multicenter Evaluation of Independent High-Throughput and RT-qPCR Technologies for the Development of Analytical Workflows for Circulating miRNA Analysis.

Author(s): Babayan A, Neumann MHD, Herdean A, Shaffer JM, Janning M, Kobus F, Loges S, Di Pasquale F, Kubista M, Schlumpberger M, Lampignano R, Krahn T, Schlange T, Sprenger-Haussels M, Pantel K, Kloten V

Publication: Cancers (Basel), 2020, Vol. 12, Page

PubMed ID: 32380788 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare cfmiRNA and EVmiRNA expression profiles obtained using next-generation sequencing (QIAseq) and two hybridization platforms (Nanostring and Toray 3D). Twenty-six miRNAs were selected for analysis of differential expression in non-small cell lung cancer patient specimens using the aforementioned platforms and two real-time PCR platforms (miRCURY and two-tailed). Blood was collected from 27 patients with NCSLC and 20 healthy controls into K₃EDTA S-Monovette tubes. Plasma was obtained within 2 h of collection by centrifugation at 2500 x g for 10 min followed by recentrifugation of the supernatant at 2500 x g for 10 min. Plasma was stored frozen at -80°C until extraction. Plasma was thawed in a 37°C water bath, vortexed, and cfmiRNA was extracted using the miRNeasy Serum/Plasma Advanced and EVmiRNA was obtained using the ExoRNeasy Maxi Kit. Extracted cfmiRNA and EVmiRNA were stored frozen in 96 well plates at -80°C. Libraries were prepared using QIAseq miRNA Library Kit and used for next-generation sequencing. cfmiRNA and EVmiRNA were quantified using the nCounter Human v3 miRNA Panel and the Toray Human miRNA Microarray ver.21 chip. miRNA was reverse-transcribed using the miRCURY LNA RT Kit and 26 selected miRNAs were quantified using miRCURY LNA SYBR Green PCR Kit. Two-tailed qPCR was used to quantify the same 26 miRNAs after reverse-transcription using TATAA GrandScript cDNA FreePrime Kit using TATAA SYBR GenMaster Mix.

   Summary of Findings:

   The highest cfmiRNA and EVmiRNA read counts in plasma from healthy individuals and those with NSCLC were obtained using QIAseq followed by Toray 3D with the lowest read counts using Nanostring. The QIAseq read counts were significantly higher for EVmiRNA in patients with NSCLC than for cfmiRNA in healthy patients and those with NSCLC (P<0.001, both). The Nanosting read counts for cfmiRNA were significantly higher in NSCLC patients than healthy controls (P<0.05). Compared to healthy controls, patients with NCSLC had 15.9%, 142.1%, and 317.6% more cfmiRNA and 162.9%, 46.3%, and 163% more EVmiRNA using QIAseq, Toray 3D, and Nanostring; respectively.

   Detection of 26 selected miRNAs was generally low, with only 15%, 35%, and 50% found in the EVmiRNA and 77%, 23%, and 31% in the cfmiRNA fractions using QIAseq, nCounter, and Nanostring; respectively.  Differential expression of these 26 miRNAs between NSCLC patients and healthy controls occurred for 2 EVmiRNAs and 11 cfmiRNAs when using QIAseq, 3 EVmiRNAs and 1 cfmiRNAs using nCounter, and 1 EVmiRNA and 1 cfmiRNAs using Toray 3D. Of these significantly differentially expressed miRNAs, only one (miR-122-5p in cfmiRNA) was significant using multiple platforms (QIAseq and nCounter, not detected using Toray 3D). Validation using miRCURY and qPCR identified differential expression in NSCLC and healthy controls for 12 and six EVmiRNAs, respectively; of which five were common and five and seven cfmiRNAs, respectively; of which three were common. Importantly, the three cfmiRNAs which were found to be differentially expressed using miRCURY qPCR and two-tailed qPCR (miR-451a, miR-16-5p, and miR-186-5p) were also differentially expressed in EVmiRNA using both platforms. Of these, miR-16-5p and miR-186-5p were also found to be differentially expressed in NSCLC versus control cfmiRNA using QIAseq but were not identified as significantly differentially expressed using the other platforms or in EVmiRNA. Further investigation showed only miR-16-5p to be upregulated >2 fold using both miRCURY qPCR and two-tailed qPCR platforms in both EVmiRNA and cfmiRNA. Interestingly, miR-21-5p was found to be upregulated using qPCR but downregulated using miRCURY qPCR.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	DNA microarray
   RNA	Real-time qRT-PCR
   RNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Extracellular vesicles
   Next generation sequencing Specific	Technology platform	Nanostring Toray 3D array
   Real-time qRT-PCR Specific	Technology platform	QIAseq NGS Nanostring Toray 3D array miRCURY qPCR Two-tailed qPCR

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