Pre-Analytical Modification of Serum miRNAs: Diagnostic Reliability of Serum miRNAs in Hemolytic Diseases

Abstract

Circulating microRNAs (miRNAs) are useful biomarkers of hemolysis. Since blood cells are the main origins of circulating miRNAs, we evaluated blood cell-related pre-analytical modification of the miRNA signatures during blood drawing and serum processing. The levels of miRNA before and after ex vivo blood drawing were analyzed with the reverse transcriptase-based polymerase chain reaction method. Furthermore, the changes of miRNA signatures caused by different time-lag between blood drawing and serum preparation by 24 h were evaluated. Finally, we compared the miRNA levels between leftover samples and samples of hemolytic diseases. Blood drawing procedure induced increments of red blood cell (RBC)-related miRNAs (miR-451a, miR-486) about 2-fold. One hour standing of blood samples before serum separation induced almost the same increases in RBC-related miRNAs. To test the clinical usefulness of miR-451a as a biomarker of hemolytic diseases, we analyzed miRNAs of samples from 10 normal subjects, 30 leftover samples in the clinical laboratory, and 20 samples from patients with hemolytic diseases. Serum miR-451a significantly increased in patients with hemolytic anemia more than the levels of pre-analytical modification. In conclusion, the pre-analytical modification of serum miRNAs did not disturb the usefulness of RBC-derived miRNAs as biomarkers of hemolytic diseases.

Diagnosis of type 2 Diabetes Mellitus (T2DM) using Paired microRNAs

“…Serum samples were obtained after blood coagulation at 25 °C for 30 min, by centrifugation of 3,500 rpm for 10 min at room temperature. After passing membrane filter with 0.45 μm pore size, samples were kept in freezers at -80 °C until use [29,30].…”

“…Finally, 30μL RNase-free-water was added onto the column and RNA solution was obtained by centrifuging at 11000 rpm for 1 minutes. RNA specimens were kept in -80°C in freezer [29,30].…”

“…Finally, 30µL RNase-free-water was added onto the column and RNA solution was obtained by centrifuging at 11000 rpm for 1 minutes. RNA specimens were kept in -80℃ in freezer [29,30]. miRNA microarray analysis For analysis of serum miRNA signatures 5 serum samples were pooled from healthy subject and T2DM patients whose HbA1c levels were 8.4-9.7%.…”

“…Next, mix 17.67 µL Master mix, 1 µL primer according to target miRNA and 1.33 µL template made in RT step in new tube and spin down after tapping. The qPCR condition was 95℃ for 10 mints, and 40 cycles of 95℃ in 15 sec, 60℃ for 60 sec [29,32]. The 2 -ΔΔCT Method is a major method of quantitate miRNA analysis to compare specific miRNAs at healthy and T2DM.…”

“…It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 30, 2022. ; https://doi.org/10.1101/2022.09. 29.510072 doi: bioRxiv preprint of patients with T2DM while 9.4% of non-T2DM persons and was the important cause of increased mortality in T2DM [13]. Quality of life (QOL) of T2DM patients is also disturbed by other complications than CVD or KD, namely infectious diseases, cancers, retinopathy, or neuropathy [1,2,14,15].…”