Sybr green master

Total RNA was isolated from healing skin, HDFs, and UCB-MSC-exo using TRIzol reagent (Sangon Biotech Co., Ltd., Shanghai, China). cDNA was synthesized from total RNA using the cDNA Synthesis Kit (Takara, Japan). qRT-PCR analysis was performed using SYBR Green Master (Roche, Switzerland) in an ABI 9700 Detection System (Thermo Fisher Scientific, MA, USA). GAPDH mRNA was used as an internal control. qRT-PCR for miRNA was performed using the Bulge-Loop^TM miRNA qRT-PCR Starter Kit (Ribobio, China) according to the manufacturer’s instructions. U6 small RNA was used as an internal control. Table S2 lists the primers used. All experiments were repeated in triplicate.

Total RNA was extracted from lung tissue with TRIzol (Life Technologies). cDNA synthesis was carried out with a Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific), according to the manufacturer's protocol. Quantitative real‐time PCR was performed using SYBR Green Master (Roche) under an ABI 7500 Sequence Detection System (Thermo). The primer sequences were listed in Table 1.

Standards for the qPCR assays were prepared from the genomic DNA of E. coli DH5a (Takara Bioengineering Co., Ltd., Dalian, China). Seven point standard curves were generated by a 10-fold serial dilution of plasmids carrying 10⁸–10² copy number of the target genes, and qPCR was performed using SYBR Green Master (Roche Diagnostics GmbH, Cambridge, MA, USA) and an ViiA™7 Dx Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with cycling conditions of 95 °C for 10 min, followed by 40 cycles of 30 s at 95 °C and 60 s at 60 °C. All standard curves had R2 values above 0.99.

Samples were prepared and analyzed as described (11 (link)). Briefly, RNA was harvested using TriPure (11667157001; Roche) and cDNA synthesized using 2-μg RNA was reverse transcribing using MMLV-RT (#28025013). qRT-PCR reactions were performed on the Lightcycler480 instrument using SYBR Green Master (Roche) with standard cycling procedures and relative quantification of mRNA levels was calculated relative to the standard curve and 18S housekeeping gene. Primer sequences for 18S, BCL2, CAIX, CXCL8, VEGFA have been reported (11 (link)). Other primer sequences are available in Supplementary Methods.

MDCK cells were harvested in TRIzol (Invitrogen) for total RNA extraction, and RNA was reverse-transcribed with a PrimeScript RT reagent kit with a gDNA Eraser (TaKaRa, Kusatsu, Japan). Quantitative real-time PCR (qRT-PCR) analyses were performed with SYBR Green Master (ROX; Roche, Indianapolis, IN, USA) in an ABI 7500 Fast thermal cycler and were analyzed with 7500 Software v2.0.1. All expression levels are reported relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PCR oligo sequences are listed as follows:
Par3
For:
GCAGTGACCCGGCTTTAATT; Rev:
CAGCAGTGTTTTGCTTCAG
Ankrd1
For:
AGCCCAGATCGAATTCCGCG; Rev:
CTCCTTCTCGGTCTTTGGCAT
Anln
For:
ATGTCTTCGTGGCCGATTTGA; Rev:
CTCTGACAGTGAGTTTCCTGTTT
Cyr61
For:
TGAAGCGCCTCCCAGTTTT; Rev:
CGGGTCTCCTTCACCAGGCG
Ctgf
For:
ACCGACTGGAAGACACGTTTG; Rev:
CCAGGTCAGCTTCACAAGG
Diaph1
For:
GCGGTATGCATTGTAGGGGA; Rev:
CAGGAGATGTAACCAGGGCA
Inhba
For:
GCTGCACGTAGGCAAAGTCG; Rev:
GCTGTGCCTGATTCCGCGAA
GAPDH
For:
ACGGATTTGGCCGTATTGGGC; Rev:
TTGACTGTGCCGTGGAATTTG

Total RNA was extracted using TRIzol reagent (Invitrogen, Paisley, UK) according to standard protocols, and RNA was quantified using a NanoDrop ND-1000 spectrophotometer (Labtech, Uckfield, UK). cDNA was synthesised using random hexamer primers and SuperScript Reverse Transcriptase (Invitrogen), according to manufacturer’s instructions. Quantitative PCR (qPCR) was performed with a Roche LightCycler 480 using SYBR Green Master (Roche), according to standard protocols. Expression of genes of interest was normalised to expression of Hprt1. Primer sequences are listed in Supplementary Table 3.

Total RNA was extracted from cell populations using an RNeasy Mini kit (Qiagen, Germantown, MD, USA) and was then transcribed into cDNA using a first-strand cDNA Kit (Takara, Osaka, Japan) according to the manufacturer’s instructions. Gene expression was measured by real-time q-RT-PCR analysis using a LightCycler instrument (Roche Diagnostics, Tokyo, Japan) with SYBR Green Master (Roche). Primers of ORF-1 and ORF-2 were synthesized as previous described [31 (link)]. The other primers were purchased from Qiagen. Expression levels were normalized to the expression of Rplp0 or β2M.