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Comparison of RNA isolation and associated methods for extracellular RNA detection by high-throughput quantitative polymerase chain reaction.

Author(s): Tanriverdi K, Kucukural A, Mikhalev E, Tanriverdi SE, Lee R, Ambros VR, Freedman JE

Publication: Anal Biochem, 2016, Vol. 501, Page 66-74

PubMed ID: 26969789 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study examined extraction of RNA from plasma using four different methods and compared yields and miRNA CT­ values. Blood was collected by venipuncture from three healthy males and three healthy females into sodium citrate tubes. Specimens were centrifuged immediately at 2000 x g for 10 min and plasma was transferred to microcentrifuge tubes and centrifuged again at 8000 x g for 10 min. The resultant supernatant was pooled in new tubes, aliquoted, and stored at -80˚C until RNA isolation. RNA was isolated using the miRCURY RNA isolation kit, the miRNeasy serum/plasma kit, the TaqMan miRNA ABC purification kit, and an in-house method based on phenol/chloroform extraction. RNA quality was assessed by the Agilent RNA 6000 Pico Kit and the Agilent Small RNA Kit on a bioanalyzer. RNA was reverse transcribed using miScript II RT Kit, preamplified using the miScript Microfluidics PreAMP kit, and quantified using 90 miScript miRNA real-time PCR assays.

   Summary of Findings:

   The Agilent Small RNA kit was unable to quantify RNA in any of the specimens. Using the Agilent RNA 6000 Pico Kit, the highest RNA yields were obtained using the miRNeasy Serum/Plasma kit (9.20 ± 3.12 ng) followed by the miRCURY RNA Isolation kit (7.36 ± 2.07 ng) and much lower yields obtained using the in-house method (1.06 ± 0.44 ng) and TaqMan miRNA ABC kit (0.70 ± 0.26 ng).  In contrast, the lowest average cycle threshold (CT) values were obtained using the in-house method (17.25 ± 0.41) and TaqMan miRNA ABC kit (17.27 ± 1.135) followed by the miRCURY RNA isolation (18.20 ± 0.955) and miRNEasy Serum/Plasma kit (19.17 ± 0.73). The coefficient of variance for each of the three replicates for the 90 gene panel was lower when extraction was with the in-house method (1.86%) than when the miRNEasy (3.77%), miRCURY (5.10%), or TaqMan (6.75%) kits were used. Further, the lowest cumulative distribution of CT values was found when the in-house method was used for extraction and the highest when the miRNEasy kit was used. Importantly, the CT values were strongly correlated among all of the isolation kits (ρ=0.88-0.96).

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	miRCURY RNA isolation kit miRNeasy serum/plasma kit TaqMan miRNA ABC purification kit In-house method

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2. Study Purpose

   This study compared CT values between serum and plasma and investigated the effects of using the miScript versus TaqMan kit for reverse transcription, preamplification, and quantification of miRNA. Blood was collected by venipuncture from three healthy males and three healthy females into sodium citrate and serum tubes. Citrated specimens were centrifuged immediately at 2000 x g for 10 min and serum tubes were allowed to clot for 30 min and centrifuged at 2000 x g for 10 min. Plasma and serum were transferred to microcentrifuge tubes, centrifuged again at 8000 x g for 10 min, and the supernatants were pooled in new tubes and then aliquoted. Pooled serum and plasma aliquots were stored at -80˚C until RNA isolation using an in-house method based on phenol/chloroform extraction. RNA quality was assessed by the Agilent RNA 6000 Pico Kit on a bioanalyzer. RNA was reverse transcribed using miScript II RT Kit and TaqMan MicroRNA RT kit. cDNA was preamplified using the miScript Microfluidics PreAMP kit and the TaqMan PreAMP kit. miRNAs were quantified using 90 miScript miRNA or TaqMan real-time PCR assays.

   Summary of Findings:

   The mean CT values were lower for serum than plasma using either the miScript RT kit (18.05 versus 19.29) or the TaqMan MicroRNA RT kit (21.25 versus 22.40) for reverse transcription. Preamplification with the miScript microfluidics kit resulted in lower mean CT values than the TaqMan PreAMP Master Mix for plasma (19.11-19.51 and 22.17-22.63, respectively) and serum (18.01-18.11 and 20.82-21.58, respectively). The correlations between replicates were higher when the miScript miRNA assays were used rather than the TaqMan miRNA assays for plasma (ρ=0.88-0.97 versus ρ=0.31-0.94) and serum (ρ=0.97-0.99 versus ρ=0.77-0.97). Finally, the distribution of CT values differed among the assays and specimen types with the miScript miRNA assay having distributions of 6-27 for plasma and 5-27 for serum and the TaqMan miRNA assay having distributions of 14-27 for plasma and 7-27 for serum.

   Biospecimens

   • Bodily Fluid - Serum
   • 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)
   Biospecimen Aliquots and Components	Blood and blood products	Serum Plasma
   Real-time qRT-PCR Specific	Template modification	Reverse transcribed with miScript Preamplified with miScript kit Preamplified with TaqMan kit Reverse transcribed with TaqMan kit
   Real-time qRT-PCR Specific	Technology platform	TaqMan assays miScript assays

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