Assessing an improved protocol for plasma microRNA extraction.
Author(s): Moret I, Sánchez-Izquierdo D, Iborra M, Tortosa L, Navarro-Puche A, Nos P, Cervera J, Beltrán B
Publication: PLoS One, 2013, Vol. 8, Page e82753
PubMed ID: 24376572 PubMed Review Paper? No
Purpose of Paper
This paper compared RNA yield and purity in microRNA (miRNA, miR) isolated from fresh and frozen plasma using three different methods. The effects of adding two different concentrations of carrier RNA prior to extraction with the mirVana Kit on RNA yield and the miRNA profile as determined by microarray was also investigated.
Conclusion of Paper
The RNA yield was higher from frozen than fresh plasma with each of the kits tested. The authors state this is likely due to differences in the centrifugation procedures used. Purity (OD 260/280) of RNA from frozen specimens tended to be lower than in fresh specimens. Although a larger 260 nm peak was observed when extraction was with Trizol than by the other methods, this was also accompanied by a much larger peak at 230 nm, indicating a high degree of organic contaminants not found using the other methods and was not improved by subsequent purification using the mirVana spin columns. The RNA concentration was higher when extraction was with the miRNeasy protocol rather than mirVana. Carrier RNA made it difficult to quantify the RNA when used at the higher concentration (10 µg/mL) but improved quantification of miRNA when used at the lower concentration (1 µg/mL). The microarray profiles of plasma without carrier or with low concentration of carrier RNA co-clustered in PCA and hierarchical clustering but the low concentration of carrier RNA increased the signal. In contrast, the profile was notably different when the standard 10 µg/mL carrier was used. While biopsy RNA did not co-cluster with plasma RNA, the authors report it was more similar to that extracted from plasma with standard concentration carrier RNA. Finally, 1496 miRNAs were found in plasma isolated without carrier RNA and with low concentration carrier RNA but only 992 were found in plasma isolated without carrier and with standard concentration carrier RNA.
Studies
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Study Purpose
This study compared RNA yield and purity in miRNA isolated from fresh and frozen plasma using three different methods. The effects of adding two different concentrations of carrier RNA prior to extraction with the mirVAna Kit on RNA yield and the miRNA profile as determined by microarray was also investigated. Peripheral blood from twenty patients with inflammatory bowel disease and healthy volunteers was obtained in K3EDTA tubes after a 12 h fast. Four patients also had biopsies obtained from bowel mucosa but details of specimen handling and RNA extraction were not provided. For most experiments, blood was layered on Histopaque 1077 and centrifuged at 213 x g for 30 min after which cells and platelets were separated from plasma by centrifugation at 2375 x g for 10 min. Plasma was then aliquoted and either used immediately for RNA isolation or stored at -80°C for later isolation. RNA was extracted from plasma using the mirVana PARIS Kit, TRIzol-LS, or the miRNeasy Serum/Plasma Kit. The mirVana Kit was also used with a change in the post phenol/chloroform centrifugation step to 30 min at 4°C. The effect of obtaining plasma by direct centrifugation of blood was tested by centrifuging blood at 1700 x g for 10 min followed by 2000 x g for 10 min and used immediately for RNA extraction. The effect of carrier RNA isolation was tested by adding 1 µg/mL (low concentration) or 10 µg/mL (standard concentration) Torulla yeast RNA carrier prior to the chloroform step in the mirVana extraction. RNA yield and purity were assessed by NanoDrop and bioanalyzer. miRNA levels were quantified in four plasma specimens isolated without carrier, two plasma specimens isolated with standard concentration carrier RNA, four plasma specimens isolated with low concentration carrier RNA, and four unspecified biopsy specimens.
Summary of Findings:
Importantly, hemolysis was <0.03 g/L in all specimens, indicating it was not a confounding factor in any of the analyses. RNA yield was higher from frozen than fresh plasma with each of the kits tested which was likely due to differences in the centrifugation procedures used. Purity (OD 260/280) of RNA from frozen specimens tended to be lower than in fresh specimens but was low using all methods. Although a larger 260 nm peak was observed when extraction was with Trizol than by the other methods, this was also accompanied by a much larger peak at 230 nm, indicating a high degree of organic contaminants not found using the other methods. The authors report subsequent purification of Trizol-isolated RNA using the mirVana spin columns did not result in purity comparable to that obtained using the mirVana Kit alone. The RNA concentration was 3-fold higher when extraction was with the miRNeasy protocol rather than mirVana alone (P<0.001). Adding carrier RNA resulted in a much larger peak at 260, indicating increased purity as well as yield, but this peak also reflected the carrier RNA. Carrier RNA made it difficult to quantify the RNA when used at the higher concentration (10 µg/mL) but improved quantification of miRNA when used at the lower concentration. The microarray profiles of plasma isolated without carrier or with low concentration of carrier RNA co-clustered in PCA and hierarchical clustering, but the low concentration of carrier RNA increased the signal. In contrast, profiles were notably different when the standard 10 µg/mL carrier was used While biopsy RNA did not co-cluster with plasma RNA, the authors report it was more similar to that extracted from plasma with standard concentration carrier RNA. Finally, 1496 miRNAs were found in plasma isolated without carrier and with low concentration carrier RNA but only 992 were found in plasma isolated without carrier RNA and with standard concentration carrier RNA.
Biospecimens
Preservative Types
- None (Fresh)
- Frozen
Diagnoses:
- Normal
- Irritable Bowel Syndrome
Platform:
Analyte Technology Platform RNA Spectrophotometry RNA Automated electrophoresis/Bioanalyzer RNA DNA microarray Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation None (fresh)
Frozen
Analyte Extraction and Purification Analyte isolation method mirVana PARIS kit
miRNeasy Serum/Plasma Kit
TRIzol-LS
TRIzol-LS and the miRNeasy Serum/Plasma Kit
No carrier RNA added
10 µg/mL carrier RNA added
1 µg/mL carrier RNA added