The Influence of Pre-analytical Factors on the Analysis of Circulating MicroRNA.
Author(s): Shiotsu H, Okada K, Shibuta T, Kobayashi Y, Shirahama S, Kuroki C, Ueda S, Ohkuma M, Ikeda K, Ando Y, Matsui H, Kayamori Y, Umemura T
Publication: Microrna, 2018, Vol. , Page
PubMed ID: 29984665 PubMed Review Paper? No
Purpose of Paper
This paper compared microRNA (miRNA, miR) levels in plasma and serum and investigated the effects of centrifugation speed, coagulation activators, filtration, RNAse inhibitors, and normalization method on miRNA levels.
Conclusion of Paper
Plasma had higher levels of miR-16 and lower levels of miR-451 compared to matched serum when blood specimens were centrifuged at 1,900 x g for 10 min; however, levels of miR-16, miR-126, and miR-223 were comparable and miR-451 levels were slightly higher in plasma when specimens were obtained by centrifugation at 10,000 x g for 1 min. Levels of endogenous miRNA were comparable in plasma incubated in tubes containing coagulation accelerators and plain tubes but levels of cel-miR-39 were 1.12-fold higher in the tube with coagulation accelerators. Levels of all four miRNAs were comparable in plasma and serum when normalized with cel-miR-39 and miR-16. Filtration of plasma with a 1 µM filter resulted in higher miR-451 levels than in unfiltered plasma, but filtration with a 0.1 µM filter resulted in lower miR-451 levels. Mixing of filtered and unfiltered plasma resulted in similar levels of miR-451 as unfiltered plasma, indicating that filtration removed inhibitory particles. The addition of RNAse inhibitor stabilized the ratio of miR-16 in plasma to serum, but had no effect on miR-451, miR-126, and miR-223 which were unaffected by storage.
Studies
-
Study Purpose
This study compared miRNA levels in plasma and serum and investigated the effects of centrifugation speed, coagulation activators, filtration, RNAse inhibitors, and normalization method. Fasting blood specimens were collected from 25 healthy volunteers into plasma tubes containing Na2EDTA and serum tubes containing coagulation activators (CAs). Plasma and serum were obtained by centrifugation at 1900 x g for 10 min or 10,000 x g for 1 min. To investigate the effects of coagulation activators contained in serum tubes, plasma was stored in plain tubes and serum tubes with CAs for 30 min before miRNA extraction. Plasma and serum were mixed with Nucleospin lysis buffer and frozen at -80˚C within 1 h of blood draw. miRNA was extracted from all specimens using the Nucleospin miRNA Plasma extraction kit. miRNAs were reverse transcribed using the High Capacity cDNA Archive kit and quantified using the TaqMan miRNA assay kits. miRNA levels were normalized to cel-miR-39 which was spiked into the specimens after homogenization.
Summary of Findings:
Plasma from four donors had higher levels of miR-16 (17.5 fold, P<0.05), lower levels of miR-451 (0.50 fold, P<0.05), and a trend toward higher levels of miR-126 (93.9 fold, P=0.058) and miR-223 (32.2 fold, P=0.08) compared to matched serum when centrifuged at 1,900 x g for 10 min; however, levels of miR-16, miR-126, and miR-223 were comparable (0.76-3.2 fold) and miR-451 levels were only slightly higher in plasma (3.83 fold, P<0.05) when plasma and serum were obtained by centrifugation at 10,000 x g for 1 min of blood from all 25 donors. Levels of endogenous miRNA were comparable in serum incubated in tubes containing coagulation accelerators and plain tubes but levels of cel-miR-39 were 1.12-fold higher in the tube with coagulation accelerators. Mean CT of cel-miR-39 (28.4 and 26.9, respectively), cel-miR-39 normalized levels of all four miRNAs (0.43 to 1.38-fold), and CVs of the CT values (11% in plasma and 8% in serum) were comparable in plasma and serum specimens. Plasma levels of miR-451, miR-126, and miR-223 were not significantly different after normalization to miR-16 (4.31, 1.21, and 1.78-fold, respectively. Compared to unfiltered plasma, miR-451 levels were higher with filtration of plasma using a 1 µM filter (1.44-fold, P=0.04) and trended higher when filtrated using a 0.45 µM filter (1.36 -fold), but miR-451 levels were lower in plasma filtered with a 0.1 µM filter (0.13-fold, P=0.03) and trended lower when filtered with a 0.22 µM filter (0.33-fold). Mixing of filtered and unfiltered plasma resulted in similar levels of miR-451 as unfiltered plasma indicating that filtration removed inhibitory particles. The authors report that serum cel-miR-39 levels decreased to 0.41-fold of initial levels after 2 h at room temperature. The addition of RNAse inhibitor stabilized miR-16 but had no effect on miR-451, miR-126, or miR-223 levels which were unaffected by storage.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
Biospecimen Aliquots and Components Centrifugation Multiple durations compared
Multiple speeds compared
Real-time qRT-PCR Specific Targeted nucleic acid miR-451
miR-16
miR-126
miR-223
cel-miR-39
Analyte Extraction and Purification RNase inactivation RNAse inhibitor added
No RNAse inhibitor added
Storage Storage conditions Plain tube
Tube with coagulation inhibitor
Biospecimen Aliquots and Components Filtration 1 µM
0.45 µM
0.22 µM
0.1 µM
Real-time qRT-PCR Specific Data handling Normalized to cel-miR-39
Normalized to miR-16