Quantstudio qpcr instrument

RNA was extracted from the apical region of heart samples using TRIzol Reagent (Thermo Fisher Scientific) according to manufacturer specifications. RNA was converted to cDNA using the High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific). Amplification reactions used the SYBR Green PCR master mix in a QuantStudio qPCR instrument (both from Thermo Fisher Scientific). Samples were run in duplicates. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) gene was used as reference transcript and conditions are listed in Supplementary Table 1. Significance was assessed by 2^−ΔΔCT method.

To analyze the expression of target genes and miRNA precursors, total RNA (1.5 μg) was subjected to DNase treatment (EN0525, Thermo Scientific™), followed by reverse transcription using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific™) according to the manufacturer’s instructions for use with oligo-dT. cDNAs were amplified by conventional end-point RT-PCR using specific primers to assess for sequence specificity. Then, real-time PCR was performed as described previously (Bustamante et al., 2018 (link)). All analyses were done in triplicate on a QuantStudio qPCR instrument (Thermo Scientific™) using a standard protocol. The efficiency of PCR amplification was derived from a standard curve generated by four 10-fold serial dilution points of cDNA obtained from a mix of all the samples. Relative RNA expression was quantified by the comparative ΔΔC_T method (Livak and Schmittgen, 2001 (link)) and normalized to the geometric mean of Profilin (NM_001297545.1) expression. The statistical significance of the observed differences was evaluated by the paired t-test. Primers used for miRNA-targets amplification and profiling were described previously (Sanz-Carbonell et al., 2019 (link)). Primers used to analyze miRNA precursors are detailed in Supplementary Table 9.

Quantification of five selected sRNAs was performed from nasopharyngeal samples corresponding to 3 non-infected and 3 SARS-CoV-2 infected patients (Supplementary Table 10), starting from small RNA ( < 200 nt) enriched fractions using REALTOTAL microRNA Kit (RBMER14, Durviz) according to the manufacturer’s instructions. Stem-loop-specific reverse transcription for miRNAs detection was performed as previously described in ref. ^{85 (link)} using a RevertAid cDNA Synthesis Kit (Thermo Scientific). All analyses were done in triplicate on a QuantStudio qPCR instrument (Thermo Scientific™) using a standard protocol. Relative RNA expression was quantified by the comparative ΔΔC_T method^{86 (link)} and normalized to the geometric mean of the small-nucleolar RNAs RNU48 (AN X96648.1), a reference gene commonly used for miRNAs estimation by RT-qPCR in humans^{87 (link)}. The statistical significance of the observed differences was evaluated by the paired t-test. Primers used for amplification assays are detailed in the Supplementary Table 11.