Mirna 1st strand cdna synthesis kit

Reverse transcription was performed using miRNA 1st-Strand cDNA Synthesis Kit (Agilent Technologies) and qPCR reactions were made with High-Specificity miRNA QPCR Core Kit (Agilent Technologies) and forward specific primers for each miRNA investigated. Human U6 RNA forward primer (Agilent Technologies) was used as normalization control. All the experiments were done in four independent replicas for each time point and sample group. The qPCR reaction was performed in 7500 Real-Time PCR System (Applied Biosystems). The cycling parameters were set for standard SYBR Green method according to manufacturer’s instructions as follow: 95°C– 10 min and 95°C– 10 sec, 60°C– 15 sec, 72°C– 20 sec for 40 cycles. The miRNA forward primers sequences are depicted in S1 Table. Statistical analysis was performed using non-parametrical Mann-Whitney tests.

Cells were harvested by centrifugation at 3500 rpm for 5 min. Total RNA was extracted using the GENEzol™ reagent (New England Biolab, Inc., Ipswich, MA, USA). For miRNA, RNA was extracted using Direct-zol RNA Miniprep Kits (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. RNA concentration was measured by the Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA). The Thermo Scientific RevertAid first-strand cDNA synthesis kit (Thermo Scientific, Waltham, MA, USA) was used to synthesize cDNA for investigating mRNA expression while the miRNA 1st-strand cDNA Synthesis Kit (Agilent Technologies, Santa Clara, CA, USA) was used to synthesize cDNA for miRNA expression. The primers were designed from literature reviews and their quality was determined by the Primer-BLAST^{57 (link)} and the BLASTN database^{58 (link)}. The primer sequences used in this study are showed in Supplement 2. Then, qPCR was performed using the designed primers, Luna^® Universal qPCR Master Mix (New England Biolab, Inc., Ipswich, MA, USA) and CFX Connect Real-Time PCR Detection System (Bio-Rad, Inc., Hercules, CA, USA). The relative mRNA and miRNA expression of target genes were analyzed by Bio-ad CFX Manager software (Bio-Rad, Inc., Hercules, CA, USA) using the 2^−∆∆CT method^{59 (link)}. GAPDH was used to normalize mRNA expression and U6 was used to normalize miRNA expression.

Reverse transcription for mRNA detection was performed using iScript Reverse Transcription Supermix (Bio-Rad, 1708841) per manufacturer’s instructions. Reverse transcription for miRNA (except for LNA probes, see below), U6 snRNA, snoRNA234, or 5.8S rRNA detection was performed using the miRNA 1st-Strand cDNA Synthesis Kit (Agilent, 600036). qPCR was performed using the SYBR Green PCR Master Mix (Thermo Fisher, 4309155) and a C1000 Touch Thermal Cycler (Bio-Rad). Primer sequences are provided in Table 2.
For locked nucleic acid (LNA) RT-qPCR, reverse transcription was performed using miRCURY LNA RT Kit (Qiagen, 339340). qPCR was performed using miRCURY LNA SYBR Green PCR Kit (Qiagen, 339346) and a C1000 Touch Thermal Cycler. MiR-338-3p was detected using the hsa-miR-338-3p miRCURY LNA miRNA PCR Assay (GeneGlobe ID: YP00204719).
For RT-qPCR experiments, data were analyzed using the 2^−ΔΔCq method [previously known as the 2^−ΔΔCt method, first described in the Applied Biosystems User Bulletin 2 (P/N 4303859)] (Livak and Schmittgen, 2001 (link)).

300 ng of each RNA sample was polyadenylated and reverse-transcribed using miRNA 1^st-Strand cDNA Synthesis Kit (Agilent Technologies). cDNA was further diluted 1:2 with RNase-free water prior to quantification by qRT-PCR (quantitative real-time PCR). Relative expression of particular miRNAs was quantified using miRNAs forward primers (Agilent Technologies) and Universal Reverse Primer (Agilent Technologies) in quantitative RT-PCR. qRT-PCR reactions were conducted using LightCycler 480 System (Roche). miRNA expression was normalized using 18S rRNA. 25 μl reaction mixture was prepared with the DyNAmo HS SYBR Green qPCR Kit (Finnzymes) and included 1× MasterMix, 0.3× ROX reference dye, 0.3 μM of each primer, 2 μl of template cDNA and water to a final volume of 25 μl. The PCR conditions for all genes were as follows: initial denaturation (95 °C, 10 min), a four-step amplification program repeated 40–50 times [95 °C for 15 s, Tm (melting temperature) for 30 s and 72 °C for 30 s], a melting curve programme (95 °C for 1 min, 55 °C for 30 s, 55–95 °C with a heating rate of 0.1 °C/s and 95 °C for 30 s). All standard curves were generated by amplifying series of 5-fold dilutions of cDNA. The quality of PCR products was checked by an analysis of the melting curve.

miRNA was extracted from the plasma of patients with COVID-19 and of the age-matched control group according to the instructions for the NucleoSpin miRNA Kit (Macherey-Nagel, Hoerdt, France) and stored at −80 °C. The concentration of isolated miRNA was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific Inc., Wilmington, DE, USA), and miRNA was reverse transcribed into cDNA using the miRNA 1st-Strand cDNA Synthesis Kit (Agilent Technologies, Lexington, MA, USA) with a universal reverse primer from the synthesis kit. Quantitative RT-PCR was conducted to detect miRNA levels using a 5× HOT FIREPolEvaGreen qPCR Mix Plus kit (no ROX) (Solis BioDyne, Tartu, Estonia) with a CFX96™ Real-Time PCR Detection System (BIO-RAD Laboratories, Inc., Singapore). For each sample, the qRT-PCR reaction consisting of a 15 min hot start at 95 °C for polymerase activation, followed by 44 cycles of 15 s at 95 °C and 20 s at 60 °C, was performed in triplicate. The 2^ΔΔCq method [93 (link)] was used for miRNA quantification analysis, with U6 as a reference. The primer sequences are listed in Table S2.