Qscript mirna cdna synthesis kit

For quantification of miRNA expression, 50 ng RNA was reverse transcribed into cDNA using qScript miRNA cDNA Synthesis kit (Quanta Biosciences), and qRT‐PCR was conducted in triplicate using Perfecta SYBR Green Super Mix (Quanta Biosciences). Thermal cycling was performed on an Applied Biosystems 7900 HT detection system (Applied Biosystems). In EVs, the relative miRNA levels were normalized to three internal controls selected from miRNA‐Seq analysis: miR‐191‐5p, miR‐26a‐5p and let‐7a‐5p, using the Delta‐Delta Ct method. For cells, fold changes were normalized to miR‐16‐5p and U6 small nuclear RNA.
For quantification of mRNA expression, 500 ng RNA was transcribed into cDNA using iScript cDNA Synthesis (Bio‐Rad), and qRT‐PCR was conducted in triplicate using Takyon MasterMix (Eurogentec). Fold changes were normalized to Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) using the Delta–Delta‐Delta Ct method. All qRT‐PCR primers are listed in Table S4.

MiRNeasy Mini Kit (QIAGEN) was used to isolate total RNA according to the manufacturer’s guidelines, and a total of 1,000 ng RNA was used as a template and then reverse transcribed to cDNA using the qScript miRNA cDNA Synthesis Kit (Quanta BioSciences, Beverly, MA). The expression level of miR-873 was measured with the PerfeCTa miRNA Assay Kit (Quanta BioSciences) using miRNA primers from Quanta BioSciences, using real-time qPCR and normalized to the level of U6 small nuclear RNA (RNU6; Quanta BioSciences), which was used as an endogenous control.
To quantify KRAS mRNA, we synthesized cDNA from the isolated RNA using a QuantiTect reverse transcription kit (QIAGEN). qRT-PCR was carried out with BX-384 Bio-Rad using the QuantiTect SYBR Green PCR kit (QIAGEN), according to the manufacturer’s protocol. All reactions were performed in triplicate. KRAS mRNA expression levels were normalized to the internal control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The sequences of the sense and anti-sense KRAS primers were 5′-ATTGTGAATGTTGGTGT-3′ and 5′-GAAGGTCTCAACTGAAATT-3′, respectively. The sequences of the sense and anti-sense GAPDH primers were 5′-CAAGGTCATCCATGA CAACTTTG-3′ and 5′-GTCCACCACCCTGTTGCTGTAG-3′, respectively. Relative quantification of mRNA and miRNA expression was calculated using the comparative threshold cycle (2^−ΔΔCt) method.

Total RNA was extracted using the miRNeasy kit (Qiagen) following the manufacturer’s protocol. Fifty nanograms of RNA was reverse transcribed into cDNA using the qScript miRNA cDNA Synthesis kit (Quanta Biosciences, Beverly, MA), and RT-qPCR was conducted in triplicate using Perfecta SYBR Green Super Mix (Quanta Biosciences). Amplification was performed on an Applied Biosystems 7900 HT detection system (Applied Biosystems, Foster City, CA). The relative miRNA levels were normalized to two internal controls SNORD 44 and SNORD 48 using the delta–delta Ct method. Primers were purchased from Integrated DNA Technologies (Coralville, IA) (listed in Supplementary Table 7).

cDNA was synthesized using qScript miRNA cDNA synthesis kit (Quanta Biosciences) according to manufacturer’s protocol and used as template for miRNA amplification using PerfeCTa miRNA assays (Quanta Biosciences) containing specific forward primers and RT² SYBR Green qPCR master mix using the LightCycler480. The expression level of target miRNAs was normalized to the expression of Snord47.

Ingenuity Pathway Analysis (IPA) software was used in order to assess diseases, biological processes, and pathway signaling associated with ARDS-related exosomal miRNAs.
Relative Quantification of miRNAs by Quantitative RT-PCR (qRT-PCR).
For quantification of miRNA expression, 50 ng RNA was reverse transcribed into cDNA using a qScript miRNA cDNA Synthesis Kit (Quanta Biosciences), and qRT-PCR was conducted in triplicate using Perfecta SYBR Green Super Mix (Quanta Biosciences). Thermal cycling was performed on an Applied Biosystems 7900 HT detection system (Applied Biosystems). Relative miRNA levels were normalized to three internal controls (miR-191-5p, miR-93-5p, and RNU-43) selected via small RNA sequencing data. The levels of these internal controls did not vary between ARDS cases and healthy controls in the validation cohort (Supplementary Figure S1).

RT-qPCR analysis was performed as described (Kato et al., 2016 (link); Kato et al., 2021 (link)). RNA was extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA). miR-379 quantification was performed using the qScript miRNA cDNA Synthesis Kit (Quanta Biosciences, Gaithersburg, MD) and amplified using PerfeCTa SYBR Green SuperMix (Quanta Biosciences). For miRNAs, specific mature miRNA sequences were used as forward primers, and the universal primer provided in the kit was used as the reverse primer. U6 was used as an internal control. A GeneAmp RNA PCR Kit (Applied Biosystems, Carlsbad, CA) and POWER SYBR Green Mix (Applied Biosystems) were used for mRNA quantification. mRNA expression was normalized to Cypa as an internal control. Sequences of primers used in this study are shown in Supplementary Table S6.