Taqman technologies

Total RNA was isolated from 100 μl of plasma using the Total RNA purification kit (Norgen Biotek Corporation). The protocol was modified adding 20 fmol of cel‐miR‐39 (Qiagen) at the lysis step as spike‐in control to monitor RNA isolation. RT‐qPCR was performed through TaqMan technologies (Thermo Fisher Scientific) following the manufacturer's protocol.
Data were normalized to cel‐miR‐39 measured in each sample, and relative expression was calculated with the delta Ct method. Nonparametric tests were used to perform statistic evaluation, using SPSS software, and p value < 0.05 was considered statistically significant.
Validation data were correlated with hematobiochemical parameters in centenarians by Spearman's correlation test performed with GraphPad Prism software v.9, and p value ≤0.05 was considered significant.

The RT-qPCR was performed through TaqMan technologies (Thermo fisher scientific, Waltham, MA, USA) following the manufacturer’s protocol. The RNA was transcribed to cDNA with the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA), and the real-time quantitative PCR (RT-qPCR) was subsequently performed with TaqMan MicroRNA Assays. The data were normalized to the cel-mir-39 measured in each sample, and the relative expression was calculated with the delta Ct method.

Total RNA was extracted from plasma-EDTA samples (100 µl) with a Total RNA Purification Kit (Norgen Biotek, Thorold, ON, Canada) according to the manufacturer’s protocol. In addition, 20 fmol of spike-in cel-miR-39 (Qiagen, Venlo, The Netherlands) was added to the plasma samples at the lysis step as control for RNA extraction efficiency. Eight miRs were chosen, having a crucial and referenced regulatory role (see Table 1), and were measured by quantitative RT-PCR in plasma samples: miR-21-5p, -126-3p, -146a-5p, -145-5p, -133a-3p, -206, -122-5p, and -363-3p. These miRs were measured by applying TaqMan technologies (Thermo Fisher Scientific, Waltham, MA, USA); this method consists of an miR-specific retrotranscription, in which RNA is first transcribed in cDNA for each miR, then cDNA is used as a template for the quantitative PCR reaction. MiR relative expression was calculated by ΔC_t method using 2 replicates for each measurement. C_t values were normalized with miR-16-5p after validation of its stability along the time series analysis (17 (link)).