Assessing sample and miRNA profile quality in serum and plasma or other biofluids.
Author(s): Blondal T, Jensby Nielsen S, Baker A, Andreasen D, Mouritzen P, Wrang Teilum M, Dahlsveen IK
Publication: Methods, 2013, Vol. 59, Page S1-6
PubMed ID: 23036329 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to identify markers of serum and plasma quality and hemolysis as well as develop a quality control system for real-time PCR analysis of cell-free microRNA (miRNA, miR).
Conclusion of Paper
The plasma specimen known to be of compromised quality had a substantially different miRNA profile than that observed in high quality specimens, even after normalization. The hemolyzed plasma and serum specimens were easily identified by a ratio of the quantification cycle (Cq) for miR-451 to miR-23a. The authors concluded a threshold of >5 indicates a moderate risk of erythrocyte contamination and a ratio of 8 would indicate a severe risk. The authors used a series of spike-in controls to monitor extraction and reverse-transcription efficiency and to identify the presence of PCR inhibitors.
Studies
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Study Purpose
The purpose of this study was to identify markers of serum and plasma quality and hemolysis as well as develop a quality control system for real-time PCR analysis of cell-free miRNA. Specimens used in the study included 381 plasma and serum specimens that passed unspecified quality control measures, one plasma specimen that was of compromised quality (likely blood contamination), and one hemolyzed plasma and serum specimen but no details of patient condition or blood collection and processing were specified. RNA was extracted from both serum and plasma specimens using a modification of the miRNeasy Mini Kit with MS2 carrier RNA and the Exiqon RNA Spike-in Kit. RNA samples were stored at -80°C. cDNA was synthesized using miRCURY LNA Universal RT cDNA Synthesis Kit and 119 miRNAs were quantified using the miRCURY LNA Universal RT microRNA PCR System and the Serum/Plasma Focus microRNA PCR Panel.
Summary of Findings:
The plasma specimen known to be of compromised quality had a substantially different miRNA profile than that observed in high quality specimens. Importantly, the compromised specimen still had a significantly different miRNA profile from the uncompromised specimens even after the data was normalized to a geometric mean. The hemolyzed plasma and serum specimens were easily identified by a ratio of the quantification cycle (Cq) for miR-451 to miR-23a of >5, which indicates a moderate risk of erythrocyte contamination. The authors concluded that a ratio of 8 would indicate a severe risk. The authors used a series of spike-in controls to monitor extraction and reverse-transcription efficiency and to identify the presence of PCR inhibitors.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Hemolysis Present
Absent