Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Quantitative PCR Measurement of miR-371a-3p and miR-372-p Is Influenced by Hemolysis.

Author(s): Myklebust MP, Rosenlund B, Gjengstø P, Bercea BS, Karlsdottir Á, Brydøy M, Dahl O

Publication: Front Genet, 2019, Vol. 10, Page 463

PubMed ID: 31191602 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to investigate the effects of hemolysis on the levels of miR-371a-3p and miR-372-3p normalized to miR-191-5p, miR-30b-5p, and/or miR-93-5p in serum. The effect of increasing hemolysate on levels of miR-93-5p, miR-20a-5p, miR-191-5p, and miR-30b-5p was also investigated. Blood was collected from two patients with testicular germ cell cancer and one healthy control into Vacutainer tubes with a clot activator. Serum was obtained by centrifugation at 2000 x g for 10 min at 20˚C after clotting for less than 1 h. Serum was aliquoted and frozen at -80˚C. Red blood cell lysates created through sonication of red blood cells from a healthy donor were added to the serum to create serial dilutions from 0-1% hemolysate in triplicate (0%, 0.008%, 0.016%, 0.03%, 0.06%, 0.125%, 0.25%, 0.5%, and 1.0%). Additionally, the RBC lysates were added to the patient serum aliquots to create five replicates per patient each of no hemolysis (0% lysate), weak hemolysis (0.05% RBC lysate), and strong hemolysis (0.5% RBC lysate). Hemolysis was determined by measuring the absorbance at 414 nm in triplicate. Total RNA was extracted using the miRNeasy kit using glycogen as a carrier. RNA was spiked with cel-miR-39-3p and reverse-transcribed using the Universal cDNA Synthesis Kit. miRNA levels were determined by real-time RT-PCR using miRCURY LNA miRNA PCR Assays. Due to the low abundance of miR-371a-3p, a 14-cycle preamplification step was included. Cq values were adjusted to UniSP4 to correct for differences in RNA extraction efficiency.

   Summary of Findings:

   Although hemolysis was first detected by visual inspection at 0.06% hemolysate, it was pronounced only after the addition of 0.25% hemolysate. As expected, the absorbance at 414 nm increased linearly with increasing RBC lysate in serum from the healthy donor and UniSP4-adjusted Cq values for miR-191-5p, miR-30b-5p, miR-93-5p, and miR-20a-5p in serum from the healthy patient decreased with increasing hemolysate content. Decreases in Cq were first noted at lower levels of hemolysate for miR-93-5p and miR-20a-5p (0.008%) than for miR-191-5p and miR-30b-5p (0.125%).

   In the patient specimens, the coefficient of variance attributed to extraction efficiency (UniSP4) was higher than that attributed to reverse transcription (cel-miR-39-3p, 3.99% versus 1.69%). miR-371a-3p and miR-372-3p were not detected in the hemolysate and thus when normalized to the hemolysis-sensitive miRs, miR-93-5p, miR-191-5p, and miR-30b-5p declined with increasing hemolysis. miR-371a-3p and miR-372-3p were significantly lower in both strong hemolysis specimens than corresponding weak hemolysis specimens (P<0.001, all) and in weak hemolysis specimens than the corresponding no hemolysis specimens (P<0.001, all) when normalized to miR-93-5p. miR-372-3p levels normalized to miR-191-5p or miR-30b-5p were significantly lower in strong rather than weak hemolysis (P<0.01, all), but differences between weak and none were patient dependent. Similarly, significance of differences in miR-371a-3p between hemolysis groups depended on the normalizer and patient. Normalization to the average of miR-191-5p or miR-30b-5p resulted in significant differences between no hemolysis and weak hemolysis for miR-371a-3p in both patients (P<0.05, both) and miR-372-3p in one patient (P<0.05) and between weak hemolysis and strong hemolysis in both patients for miR-371a-3p (P<0.05 and P<0.01) and miR-372-3p (P<0.01 and P<0.001). Normalization to the average of miR-93-5p or miR-30b-5p resulted in significant differences between no hemolysis and weak hemolysis in both patients for miR-371a-3p (P<0.01, both) and miR-372-3p (P<0.01, both) and between weak hemolysis and strong hemolysis in both patients for miR-371a-3p (P<0.05, both) and miR-372-3p (P<0.001, both).

   Biospecimens

   • Bodily Fluid - Serum

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal
   • Neoplastic - Germ Cell

   Platform:

   Analyte	Technology Platform
   Protein	Spectrophotometry
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR -93-5p miR-20a-5p miR-191-5p miR-30b-5p miR-371a-3p miR-372-3p UniSP4 cel-miR-39
   Biospecimen Aliquots and Components	Hemolysis	Hemolysate added No hemolysate added

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