The study design
Blood samples were collected from 25 healthy donors. The donors were recruited by the Andalusian Public Health System Biobank (Granada, Spain). Twelve samples from each donor were collected: four blood samples that were collected in PaxGene tubes (2,5 ml; Preanalytix, Switzerland), and eight samples in Tempus tubes (3 ml; by Life Technologies, Germany).
The potential influence that individual technicians may have over the outcome of the sample analysis was evaluated. Consequently, two technicians processed 150 blood tubes, each using the three extraction methodologies mentioned above. Duplicate samples were processed for the 25 donors (n=50), to check the reproducibility of each technicians’ with regards to the three tested extraction methodologies (n=150) (Figure 1).
RNA extraction
Three extraction methodologies were evaluated. For each donation, the RNA was extracted from four blood tubes by each methodology, so that duplicate samples could be processed for the 25 donors (n=50) by each technician.
Methodology A: PaxGene® tubes and the PaxGene® Blood miRNA Kit (Preanalytix, Switzerland). The PaxGene tubes were semi-automated processed using the Qiacube robot (Qiagen) [13].
Methodology B: Tempus® tubes and the MagMax for Stabilized Blood Tubes RNA Isolation Kit (ThermoFisher, USA) [14] and a DynaMag-2 stand (Life Technologies, USA).
Methodology C: Tempus® tubes and the Tempus™ Spin RNA Isolation Kit ( Applied Biosystems, USA) [15]. No optional DNase treatment was performed.
RNA quantification and quality analysis
The RNA yield and its purity were measured using an Infinite F200 spectrophotometer (Tecan, Switzerland). The concentration was measured against the A260 nm value, while A260/A280 nm and A260/A230 ratios were used to calculating the purity.
Forty-two samples from 7 donors were measured by fluorimetry technique using the Quant-iT™ RiboGreen™ RNA Assay Kit (ThermoFisher Scientific, EEUU) (Figure 1).
RNA integrity analysis
Samples RNA integrity was measured using the Bioanalyzer 2100 (Agilent Technologies Ind., USA) and the Agilent RNA 6000 Nano Kit (Agilent Technologies Inc.). The RNA integrity (RIN value) was calculated.
cDNA synthesis
Samples from 7 donors (n=42) were randomly selected for cDNA synthesis and expression analysis. Samples were extracted by the two technicians using the three methodologies. RNA was reverse-transcribed using miScript II RT Kit (Qiagen) (Additional File 1).
miRNA qPCR
Expression assays were generated in triplicate for three miRNAs (miR16, miR26, and miR30), with the miScript SYBR Green PCR Kit (Qiagen) (Additional File 1).
mRNA qPCR
Expression assays were generated in triplicate for three mRNAs (18S rRNA [16], ACTB [17], and GAPDH) [18]). The FastStart Essential DNA Green Master Kit (Roche LifeScience) was used for amplification (Additional File 1).
DNase treatment
An extraordinary DNase treatment was carried out on 20 µl of selected RNAs using the RNase-Free DNase Set (Qiagen).
Statistical analysis
A statistical analysis was performed using the IBM SPSS Statistics program 17.0.3 (SPSS, Inc., Chicago, IL). A descriptive statistic analysis was also performed with the XLSTAT v2017.6 program. The ANOVA test (p<0.05) and the ‘post-hoc test after ANOVA’ -Scheffé´s method- (p<0.05) was applied to compare the mean differences between methodologies. Grubbs test was applied for outliers identification. Box plots were constructed with the XLSTAT v2017.6 program for the visualisation of replicas variability.