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Multicentric Evaluation of Circulating Plasma MicroRNA Extraction Technologies for the Development of Clinically Feasible Reverse Transcription Quantitative PCR and Next-Generation Sequencing Analytical Work Flows.

Author(s): Kloten V, Neumann MHD, Di Pasquale F, Sprenger-Haussels M, Shaffer JM, Schlumpberger M, Herdean A, Betsou F, Ammerlaan W, Af Hällström T, Serkkola E, Forsman T, Lianidou E, Sjöback R, Kubista M, Bender S, Lampignano R, Krahn T, Schlange T

Publication: Clin Chem, 2019, Vol. , Page

PubMed ID: 31235535 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare miRNA extraction efficiency, NGS quality metrics, and miRNA levels among plasma specimens extracted in different locales using different methods including those that isolated exosomal miRNA. Blood from 10 healthy donors was collected by venipuncture into K₂EDTA tubes. Plasma was obtained within 4 h of blood collection by centrifugation at 1900 x g for 10 min followed by 16000 x g for 10 min. Plasma was divided into two 2 mL aliquots and shipped on dry ice. Specimens were thawed once, spiked with cel-miR-39, and aliquoted for the different extraction methods (200 µL) and refrozen. RNA was extracted using the following kits/locations: Thermo-Fisher Scientific’s mirVana miRNA Isolation Kit (University of Athens), QIAGEN’s miRNeasy Serum/Plasma Kit (IBBL), QIAGEN’s miRNeasy Advanced Serum/Plasma Kit (QIAGEN), Norgen’s Plasma/Serum RNA Purification Mini Kit (TATAA), and Exiqon’s miRCURY Biofluids Kit (Bayer AG). Additionally, exosome miRNA was extracted using QIAGEN’s exoRNeasy Serum/Plasma Kit (QIAGEN) and by filtration though a 0.8 µm syringe filter, ultracentrifugation at 100,000 x g for 2 h at 4˚C, and freezing in PBS at -80˚C followed by RNA extraction using the miRNeasy Micro Kit. Extracted RNA was stored at -80˚C until use. cDNA was generated using the miScript II RT kit. RNA was quantified by real-time RT-PCR using customized miScript assays for let-7a-5p, miR-150 –5p, miR-16 –5p, miR-122–5p, miR-21–5p, miR-191–5p, and cel-miR-39-3p. NGS libraries were constructed using a QIAseq miRNA library kit and sequenced on a NextSeq 500.

   Summary of Findings:

   The mean CT for the PCR positive control was 18.78 and the difference between the mean RT control and the mean PCR control was 3.49, indicating high quality RNA and little to no RT inhibition. When compared to the base mean (Cq=20.13), the recovery of cel-miR-39 was significantly lower using miRNeasy Serum/Plasma (mean Cq=20.87, P<0.001) or miRNeasy Advanced Serum/Plasma kits (mean Cq=21.24 P<0.00133), slightly higher using the Norgen Plasma/Serum RNA Purification Mini (mean Cq=19.78) or miRVana kits (mean Cq=20.05), and significantly higher using miRCURY Biofluids Kit (mean Cq=18.68, P<0.0001). The mean Cq of each of the target miRNAs differed by up to 3 Cq among the extraction methods/sites with significantly lower mean Cq than the average (27.59) reported using the miRCURY Biofluids Kit (mean 26.6, P<0.001) or miRNeasy Advanced Serum/Plasma Kit (mean 26.0, P<0.001).  Similarly, significantly more sequencing reads were obtained when miRNA was extracted using the miRCURY Biofluids Kit (3.73 x 10⁶) than the other methods (mean 2.89 x 10⁶, P<0.001, all).  The percentage of reads mapped was also highest when miRNA was extracted using the miRCURY Biofluids Kit (39%). Principle Component Analysis clearly separated groups by extraction method with specimens extracted using miRCURY Biofluids Kit, miRNeasy Advanced Serum/Plasma Kit, and miRNeasy Serum/Plasma Kit clustering together with exoRNeasy and specimens extracted using Plasma/Serum RNA Purification Mini and mirVana kits clustering separately. Importantly, the strongest correlation between Cq and NGS normalized counts was observed when miRNA extraction was with the miRCURY Biofluids Kit (r=-0.728)

   For the exosomal miRNA methods, recovery of cel-miR-39-3p was higher using the exoRNeasy Kit (mean Cq=17.43) than the ultracentrifugation-based method (mean Cq=18.05).  Similarly, use of the exoRNeasy kit resulted in significantly lower mean Cq=26.89 than the base mean (27.89, P=0.00196) for the six endogenous miRNAs and use of the ultracentrifugation protocol resulted in a significantly higher mean (Cq=29.0) than the base mean Cq (27.89, P<0.001). Similarly, significantly more sequencing reads were obtained when miRNA was extracted from exosomes using the exoRNeasy Kit (2.35 x 10⁶) than the ultracentrifugation method (7.65 x 10⁵, P<0.001). Use of either exosomal extraction kit resulted in a high percentage of inserts that were <16 bp (37% for exoRNeasy and 48% for ultracentrifugation). Interestingly, the authors report that Cq values for cell-free miRNA were more strongly correlated with NGS read counts, but correlation coefficients were not provided.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Thermo-Fisher Scientific’s mirVana miRNA Isolation Kit (University of Athens) QIAGEN’s miRNeasy Serum/Plasma Kit (IBBL) QIAGEN’s miRNeasy Advanced Serum/Plasma Kit (QIAGEN) Norgen’s Plasma/Serum RNA Purification Mini Kit (TATAA) Exiqon’s miRCURY Biofluids Kit (Bayer AG) QIAgen’s exoRNeasy Serum/Plasma Kit (QIAGEN) Filtration, ultracentrifugation at 100,000 x g followed by RNA extraction using the miRNeasy Micro Kit
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-21–5p miR-191–5p cel-miR-39-3p let-7a-5p miR-150 –5p miR-16 –5p
   Next generation sequencing Specific	Technology platform	Real-time PCR

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2. Study Purpose

   This study investigated the effects of normalizing real-time RT-PCR and NGS data to miR-16 or cel-miR-39 and compared results of miRNA extraction methods. Blood from 10 healthy donors was collected by venipuncture into K₂EDTA tubes. Plasma was obtained within 4 h of blood collection by centrifugation at 1900 x g for 10 min followed by 16000 x g for 10 min. Plasma was divided into two 2 mL aliquots and shipped on dry ice. Specimens were thawed once, spiked with cel-miR-39, aliquoted for the different extraction methods (200 µL), and refrozen. RNA was extracted using the following kits/locations: Thermo-Fisher Scientific’s mirVana miRNA Isolation Kit (University of Athens), QIAGEN’s miRNeasy Serum/Plasma Kit (IBBL), QIAGEN’s miRNeasy Advanced Serum/Plasma Kit (QIAGEN), Norgen’s Plasma/Serum RNA Purification Mini Kit (TATAA), Exiqon’s miRCURY Biofluids Kit (Bayer AG). Additionally, exosome miRNA was extracted using QIAgen’s exoRNeasy Serum/Plasma Kit (QIAGEN) and by filtration though a 0.8 µm syringe filter, ultracentrifugation at 100,000 x g for 2 h at 4˚C, and freezing in PBS at -80˚C followed by RNA extraction using the miRNeasy Micro Kit. Extracted RNA was stored at -80˚C until use. cDNA was generated using the miScript II RT Kit. RNA was quantified by real-time PCR using customized miScript assays for let-7a-5p, miR-150 –5p, miR-16 –5p, miR-122–5p, miR-21–5p, miR-191–5p, and cel-miR-39-3p. NGS libraries were constructed using a QIAseq miRNA library kit and sequenced on a NextSeq 500.

   Summary of Findings:

   Using both real-time PCR and NGS, miR-16 displayed the highest expression. The variability in miR-21 was decreased 32.8% when Cq values were normalized to cel-miR-39, but there was no change in variability among the other miRNAs. In contrast, normalization to miR-16 increased the variability in miRNA levels compared to when raw Cq levels were used. However, the change in standard deviation in miRNA expression after normalization to miR-16 rather than raw Cq was much higher when extraction was with the miRCURY Biofluids (24.78%), miRNeasy Serum/Plasma (27.52%), miRNeasy Advanced Serum/Plasma (32.10%), or Plasma/Serum RNA Purification Mini (39.64%) kits, than with miRvana (4.55%), exoRNeasy (0.00%), or ultracentrifugation (4.02%). In contrast, extraction method had a smaller effect on the change in standard deviation after normalization to cel-miR-39 (-6.1 to 4.09%).

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Real-time qRT-PCR Specific	Data handling	Raw Cq Normalized to cel-miR-39 Normalized to cel-miR-16
   Analyte Extraction and Purification	Analyte isolation method	Thermo-Fisher Scientific’s mirVana miRNA Isolation Kit (University of Athens) QIAGEN’s miRNeasy Serum/Plasma Kit (IBBL) QIAGEN’s miRNeasy Advanced Serum/Plasma Kit (QIAGEN) Norgen’s Plasma/Serum RNA Purification Mini Kit (TATAA) Exiqon’s miRCURY Biofluids Kit (Bayer AG) QIAgen’s exoRNeasy Serum/Plasma Kit (QIAGEN), Filtration, ultracentrifugation at 100,000 x g followed by RNA extraction using the miRNeasy Micro Kit

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