Direct zol rna isolation kit

Scramble control, shTRPV4 or shAQP5 cells were grown to 95% confluency and their total RNA was isolated using Direct-zol RNA isolation kit (Zymo Research) according to manufacturer’s recommendations. Reverse transcription and qPCR were performed using standard techniques described previously^{19 (link)} using the following primer sets: TRPV4: F, 5′-CCCGTGAGAACACCAAGTTT-3′ and R, 5′-GTGTCCTCATCCGTCACCTC-3′; AQP5: F, 5′-CAGCTGGCACTCTGCATCTT-3′ and R, 5′-TGAACCGATTCATGACCACC-3′; MIIA: F, 5′-ATCCTGGAGGACCAGAACTGCA-3′ and R, 5′-GGCGAGGCTCTTAGATTTCTCC-3′; MIIB: F, 5′-GCTGATGGCAACTCTCCGAAAC-3′) and R, 5′-CTTCCAGGACACCATTACAGCG-3′); MIIC: F, 5′-CAGCCGTCAAATGCAAACCGAG-3′ and R, 5′-TTGCCTCTGTCGTCACCTTCTC; 18S: F, 5′-CAGCCACCCGAGATTGAGCA-3′ and R, 5′-TAGTAGCGACGGGCGGTGTG-3′.

Total RNA was isolated using a Direct-zol RNA isolation kit (Zymo Research, CA, USA) and reverse transcribed using RevertAid reverse transcriptase (Thermo Fisher Scientific) following the manufacturer's protocol. Quantitative real-time PCR (qPCR) was carried out using iQ SYBR^® Green Supermix (Bio-Rad) and a CFX Connect Real-Time PCR Detection System (Bio-Rad). Relative expression was calculated by normalizing to beta-actin or gapdh as housekeeping genes. A list of primers used in this study is shown in Supplementary Table S3. The results were calculated and presented as relative quantifications using the 2^−∆∆ct method.

For qPCR, cultured epithelial cells were collected and mRNA was extracted in TRIzol and stored at −80°C. Cultures containing 3T3-J2 fibroblasts were differentially trypsinised to remove feeder cells as described above. After thawing on ice, total RNA was isolated using a Direct-zol RNA Isolation Kit (Zymo Research). RNA quantity was assessed using a Nanodrop One system and reverse transcription was performed using qScript cDNA SuperMix (Quantabio). The following Taqman pre-designed, inventoried probes, along with 2X PCR Master Mix (Applied Biosciences), were used for qPCR reactions: KRT5 (Hs00361185_m1), KRT14 (Hs00265033_m1), KRT8 (Hs01595539_g1), TP63 (Hs00978339_m1), NGFR (Hs00609977_m1), ITGA6 (Hs01041011_m1), MUC5AC (Hs01365616_m1), MUC5B (Hs00861595_m1), FOXJ1 (Hs00230964_m1) and B2M (Hs00187842_m1). qPCR was performed under standard conditions using an Eppendorf Real-Time PCR machine. Relative RNA quantitation was based on ΔCT calculations and all samples were compared to the housekeeping gene β2-microglobulin. For MCIDAS detection, primers (supplementary table S2) and Power SYBR™ Green PCR Master Mix (Thermo Fisher Scientific) were used in standard conditions and relative RNA quantification was normalised to the housekeeping gene GAPDH.