Syn cel mir 39

For mRNA extraction, 75 µl LCL suspension containing ∼5 × 10⁶ cells was taken through the QIAGEN RNeasy Mini Kit protocol following the manufacturer’s instructions, with total RNA eluted in 40 μl RNase-free water. The quantity and quality of the extracted RNA were determined using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, UK) and a 2100 Bioanalyzer (Agilent, USA), respectively. For miRNA extraction, 100 μl LCL suspension containing ∼6.6 × 10⁶ cells was taken through the Maxwell 16 miRNA tissue kit protocol using the Maxwell 16MDX instrument following the manufacturer’s instructions, with miRNA eluted in 60 μl RNase-free water. To control for variations of miRNA extraction, each sample was spiked with 5 μl of 5 nM synthetic Caenorhabditis elegans miRNA 39 (Syn-cel-miR-39) (Qiagen, UK). Following extraction, it was not possible to quantify the miRNA concentration of LCL samples using the Nanodrop system, therefore, equivalent LCL volumes were used as input for the RNA extraction. Bioanalyzer Nanochips (Agilent) were used to identify the presence of miRNAs in the extracted samples (Supplementary Fig. 1). All extracted RNA/miRNA samples were stored at −80°C prior to expression profiling.

Whole blood (2 mL) was drawn into Vacutainer (anti-coagulated with EDTA, BD, New Jersey, US), and plasma isolation was done within 2 hours by centrifugation at 1000 g for 10 minutes. Total RNA of plasma was harvested with the TRI Reagent (Sigma Aldrich, St. Louis, US). For real time PCR, an internal control of 5 fmol synthetic Syn-cel-miR-39 (Qiagen, Valencia, CA) was added to plasma after mixing with TRI Reagent, as previously described [24] (link).

miRNA isolation was performed with the miRNeasy Mini Kit (Qiagen; Cat. No. 217004), according to the manufacturer's protocol ‘Purification of Total RNA, including Small RNAs, from Animal Cells'. In order to monitor the miRNA recovery efficiency and to normalize miRNA expression in the real‐time PCR analysis, 5 μL of Syn‐cel‐miRNA‐39 miScript miRNA Mimic 5 nmol/L (Qiagen; Cat. No. MSY0000010) was spiked into each sample, before nucleic acid preparation. RNA concentration and quality were determined with NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific). Mature miRNAs were converted to cDNA with miScript II RT kit (Qiagen; Cat. No. 218161). The CFX96 Touch^™ Real‐Time PCR Detection System (Bio‐Rad Laboratories) was used to monitor miR‐1 levels (MIMAT0000416). Each reaction was carried out with SYBR Green PCR Kit (Qiagen; Cat. No. 218073) and specific miScript Primer Assays for miR‐1 (Qiagen; Cat. No. MS00012943) and Syn‐cel‐miR‐39 (Qiagen; Cat. No. MS00019789). ΔCt value was as Ct_miR‐1 − Ct_miR‐39 in order to obtain miR‐1 levels as 2^−ΔCt. Fold change was then obtained as 2^−ΔΔCt and calculated as 2^−ΔCt of the treatment group/2^−ΔCt of the control group. For fold change greater than 1, fold regulations were reported equal to the fold change values. For fold changes lower than 1, fold regulations were reported as the negative inverse of the fold change values.