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Evaluation of optimal extracellular vesicle small RNA isolation and qRT-PCR normalisation for serum and urine.

Author(s): Crossland RE, Norden J, Bibby LA, Davis J, Dickinson AM

Publication: J Immunol Methods, 2016, Vol. 429, Page 39-49

PubMed ID: 26723490 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare different methods for the isolation of EV and extraction of EV RNA from serum and urine specimens. The authors also assessed the variability associated with levels of individual small RNAs and selected the most stable as candidates for normalization using several different methods. Blood and urine were collected from 10 healthy volunteers and 5 hematopoietic stem cell transplantation (HSCT) patients into plain vacutainer tubes and universal tubes, respectively, at eight time points: 7 days before transplant, and 0, 7, 14 and 28 days and 3, 6 and 12 months after transplant.  Only specimens from three individual were used to compare EV isolation methods. Blood was allowed to clot (time not specified) before separation of serum by centrifugation at 500 g for 5 min.  Serum was stored at -80°C while urine was stored at -20°C. Serum was centrifuged at 4500 g for 15 min to remove platelets before EV isolation using the ExoQuick Exosome Precipitation Kit or the Life Technologies Total Exosome Isolation Reagent. RNA was extracted from isolated serum EVs using the Norgen Biotek Total RNA Purification Kit, the System Biosciences SeraMir Exosome RNA Purification Column Kit, the Qiagen miRNeasy Micro Kit, the Ambion mirVana miRNA Isolation Kit, and the Invitrogen Total Exosome RNA & Protein Isolation Kit. EVs were isolated from urine with the Norgen Biotek Urine Exosome RNA Isolation Kit or by ultracentrifugation; RNA was isolated from urine EVs with the Norgen Urine Exosome RNA Isolation Kit or the Norgen Total RNA Purification Kit. RNA was quantified using the 6000 Pico Kit on a Bioanalyzer. EVs were resuspended in phosphate buffered saline and analyzed by transmission electron microscopy and nanoparticle tracking analysis. Levels of HY3, RNU48, miR-320, RNU6B, RNU19, U6, RNU38B and RNU43 were quantified by TaqMan real-time RT-PCR.   Validation was retrospectively performed using serum and urine from 55 healthy individuals and 50 HSCT patients.

   Summary of Findings:

   For serum, EV isolation with Total Exosome Isolation Reagent resulted in a narrower EV size distribution and a smaller mean size compared to isolation with the ExoQuick Exosome Precipitation Solution (128 versus 142 nm, 115 versus 129 nm and 181 versus 150 nm for the three specimens, respectively), although mean EV concentrations were comparable between the two exosome isolation methods. RNA yield was higher for two of three specimens when EVs were isolated using the Total Exosome Isolation Reagent compared to the ExoQuick Kit (899/2060/3381 pg/μl versus 1099/648/1453 pg/μl, for the three specimens, respectively). EVS isolated with the Total Exosome Isolation Reagent also had lower CT values for HY3, RNU48 and U6 compared to EV isolation with the the ExoQuick Kit. When RNA was extracted from EVs isolated from serum with the Total Exosome Isolation Reagent, the authors deemed miRNeasy the best of the five RNA extraction methods evaluated as it resulted in high yields (highest yields from two specimens and the second highest from the other three) and the lowest CT values for RNU48, RNU49, and U6. The authors reported that the Invitrogen Total Exosome RNA & Protein Isolation Kit was the second-best kit for RNA extraction from EVs isolated from serum as it produced the second lowest CT values.  RNase treatment of EVs did not affect RNA yield or CT values, indicating the majority of RNA was EV-associated.

   EVs isolated from urine using the Norgen Kit and those isolated by ultracentrifugation were similar in appearance, but mean and modal sizes were slightly smaller when isolated using the Norgen Kit (191 versus 224 and 121 versus 180, respectively). RNA yield from isolated EVs was higher when they were isolated from urine using the Norgen Kit rather than by ultracentrifugation. Further, the CT values for HY3 and U6 were lower when EV isolation was with the Norgen Kit rather than ultracentrifugation, but the difference was not significant.  RNase treatment of urine EVs had no effect on RNA yield, indicating the majority of RNA was EV associated.

   When levels of individual small RNAs were assessed in ten serum specimens, the lowest variability [lowest standard deviation (SD) and coefficient of variance (CV)] were observed for miR-320 (SD=1.02, CV=4.3%) followed by HY3 (SD=1.77, CV=6.2%), RNU6B (SD=2.03, CV=5.9%), U6 (SD=2.14, CV-8.6%) RNU38B (SD=2.45, CV=6.8%), and RNU48 (SD=2.95, CV=8.7%). Normfinder and BestKeeper identified miR-320 and as the most stable controls in serum, HY3 followed by U6.  In urine, the lowest SD and (CV) were observed for miR-320 (SD=1.27, CV=4.6%) followed by HY3 (SD=1.67, CV=6.4%), RNU48 (SD=1.85, CV=5.3%) and U6 (SD=2.60, CV=9.6%). Normfinder identified miR-320 and HY3, followed by U6 and RNU48, as the most stable controls in urine while BestKeeper identified miR-320, HY3 and RNU48 as the most stable. The authors state that while miR-320 was stable, it was not a suitable choice for a control based on its potential biological role.  RNU6, while stable, was excluded for use in serum due to its low level of expression. Thus, HY3 and U6 were chosen as the best normalizers for serum and HY3 and RNU48 as the best normalizers for urine.  In the validation cohort of 55 serum specimens and 50 urine specimens, low SD and CVs were observed for HY3 in serum (2.22 and 7.4%, respectively) and urine (2.42 and 8.8%, respectively), as well as U6 in serum (2.93 and 11.8%, respectively) and RNU48 in urine (2.26 and 6.9%, respectively).

   Biospecimens

   • Bodily Fluid - Urine
   • Bodily Fluid - Serum

   Preservative Types

   • Frozen

   Diagnoses:

   • Other diagnoses
   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Light scattering
   RNA	Real-time qRT-PCR
   RNA	Automated electrophoresis/Bioanalyzer
   Morphology	Electron microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Biospecimen location	Serum Urine
   Real-time qRT-PCR Specific	Targeted nucleic acid	HY3 RNU48 RNU38B RNU6B RNU19 miR-320 U6 RNU43
   Real-time qRT-PCR Specific	Data handling	Analysis with BestKeeper Analysis with NormFinder Analysis of SD and CV
   Analyte Extraction and Purification	Analyte isolation method	Comparison of exosome isolation from serum with ExoQuick Exosome Precipitation Kit versus Life Technologies Total Exosome Isolation Reagent Comparison of exosome isolation from urine with Norgen Biotek Urine Exosome RNA Isolation Kit versus ultracentrifugation RNA extraction from serum EVs with Total RNA Purification, SeraMir, miRNeasy, mirVana and Total Exosome RNA & Protein Isolation kits

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