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Comparison of protocols and RNA carriers for plasma miRNA isolation. Unraveling RNA carrier influence on miRNA isolation.

Author(s): Ramón-Núñez LA, Martos L, Fernández-Pardo Á, Oto J, Medina P, España F, Navarro S

Publication: PLoS One, 2017, Vol. 12, Page e0187005

PubMed ID: 29077772 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared RNA and miRNA recovery and integrity among plasma specimens extracted with the miRNeasy or miRCURY kits with or without carrier RNA. The effects of miRNA sequence, abundance, and predicted protein folding as determined by the change in Gibbs free energy (ΔG) as well as normalization method (volume versus endogenous control) on miRNA recovery was also examined. Blood collected from 10 healthy patients was centrifuged at 1500 x g for 30 min at 4˚C to obtain platelet poor plasma (PPP). PPP was aliquoted and frozen at -80˚C until RNA isolation. PPP was thawed on ice and centrifuged at 3000 x g for 5 min before RNA was isolated using the phenol and column-based miRNeasy mini kit or the column-based miRCURY RNA isolation kit and stored at -80˚C. RNA was extracted from an additional 18 plasma specimens (details not provided) with or without yeast carrier RNA or MS2 bacteriophage carrier RNA using the two protocols. RNA concentration and purity were determined by spectrophotometer. RNA integrity was determined by bioanalyzer.  RNA was reverse-transcribed and amplified using the microRNA LNA primer mix.  

   Summary of Findings:

   Although the miRCURY kit resulted in higher RNA recovery when no MS2 or yeast carrier RNA were included in the extraction, the highest RNA recovery was obtained using the miRNeasy kit with MS2 RNA carrier. However, bioanalyzer analysis showed a smear for specimens extracted with the miRNeasy kit with MS2 carrier RNA and higher recovery of small miRNA when yeast carrier RNA was used with the miRNeasy kit. Based on the ratio of the absorbance at 260 to 280, Only RNA extracted with the miRNeasy kit with MS2 carrier RNA had acceptable purity (1.80 versus <1.7 for all other specimens) and A260/230 ratios were below 0.5 for all specimens. Despite the impurities, amplification of spiked-in RNA occurred with similar efficiencies in all extractions. As expected, inclusion of carrier RNA, especially yeast carrier RNA, resulted in higher recovery of the exogenous UniSp2 RNA. The highest miRNA recovery per µL of plasma as determined by real-time PCR was obtained using the miRNeasy kit with yeast carrier RNA followed by the miRCURY kit with yeast carrier RNA. Generally, the miRCURY kit yielded more miRNA per µL of plasma than the miRNeasy kit when no carrier was included, but the number of copies of let-7a-5p, miR-122-5p, and miR-320a were comparable between kits when no carrier was included.

   Interestingly, there was a weak negative correlation between the number of amplified copies of miRNA per µL of plasma extracted using miRCURY kit with yeast carrier RNA and the GC content (r=-0.290, P<0.001) and ΔG (r=-0.103, P=0.023), but not the miRNA relative abundance. The mean fold recovery of miRNA was strongly negatively correlated with GC content (r=-0.799, P<0.001) but not ΔG. When only miRNAs with a GC content of 38.64-50% were considered, there was a significant very weak correlation between fold recovery of miRNA using the miRCURY kit with yeast carrier RNA with the logarithm of miRNA relative abundance (r=0.133, P=0.026) and the miRNA ΔG (r=-0.223, P <0.001).­­ The mean fold recovery using miRCURY with yeast carrier RNA for each miRNA was not correlated with the relative abundance but was strongly negatively correlated with the ΔG (r=-0.752, P=0.002). In contrast, when RNA was extracted using miRNeasy with yeast carrier RNA, individual and mean fold recovery were significantly correlated with relative abundance (r=0.293, P<0.001 and r=0.468, P=0.021, respectively) but not with GC content or ΔG. Thus the authors conclude that the effect of yeast carrier RNA on recovery of a particular miRNA using the miRCURY kit is influenced by the GC content, the ΔG, and, to a small extent, by the relative abundance but the effect of yeast carrier RNA with the miRNeasy kit is mostly based on relative abundance.

   Normalization of miRNA levels to miR-103a-3p rather than plasma volume decreased the between-kit differences in fold abundance of miRNAs, but significant differences still existed. For instance, miR-103a-3p normalized levels of all four miRNA examined (miR-15a-5p, miR-23a-3p, miR-101-3p, and miR-320a) were significantly lower when extracted using the miRNeasy kit with carrier yeast RNA rather than miRCURY with carrier yeast RNA (P<0.001, P<0.05, P<0.001, and P<0.001, respectively) and levels of miR23a-3p were higher when extracted with either kit with yeast carrier RNA than without a carrier (P<0.05, both). Although most of the differences between protocols were between the miRNeasy and miRCURY kits when yeast carrier RNA was used, the highest correlation in recovery levels was also observed between the miRNeasy and miRCURY kits when yeast carrier RNA was included (r=0.532, P<0.001). 

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	miRNeasy kit miRNeasy kit with MS2 carrier RNA miRNeasy kit with yeast carrier RNA miRCURY kit miRCURY kit with MS2 carrier RNA miRCURY kit with yeast carrier RNA
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-103a-3p miR-15a-5p miR-23a-3p miR-101-3p miR-320a
   Real-time qRT-PCR Specific	Data handling	Normalized to plasma volume Normalized to miR-103a-3p

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