Sensimix sybr and fluorescein kit

We extracted total RNA from WT and DP-null keratinocytes with the Qiagen RNeasy mini kit and used the SuperScript First-Strand Synthesis System (Invitrogen) to generate cDNA. Quantitative-comparative RT-PCR was performed on a StepOne Plus Real-Time PCR System (Life Technologies) using SYBR green reagent (SensiMix SYBR and Fluorescein Kit; Bioline). The average of the wild-type levels was set to 1 and levels of mRNA from the mutant were relative to this. All primer sequences for claudin genes assayed were previously validated (http://pga.mgh.harvard.edu/primerbank/). Cldn1 5′-ggggacaacatcgtgaccg and 5′-aggagtcgaagactttgcact, Cldn3 5′-accaactgcgtacaagacgag and 5′-cagagccgccaacaggaaa, Cldn4 5′-gtcctgggaatctccttggc and 5′-tctgtgccgtgatgttg, Cldn5 5′-gcaaggtgtatgaatctgtgct and 5′-gtcaaggtaacaaagagtgcca, Cldn6 5′-atggcctctactggtctgcaa and 5′-gccaacagtgagtcatacacctt, Cldn8 5′-gcaacctacgctcttcaaatgg and 5′-ttcccagcggttctcaaacac, Cldn10 5′-cgaatgagaaagtgaccaccc and 5′-attagtcctctacatgcctggat, Cldn11 5′-atggtagccacttgccttcag and 5′-agttcgtccatttttcggcag, Cldn12 5′-tgtccttcctgtgtggtattgc and 5′-aaatcgtcaggttcttctcgttt, Cldn18 5′-ccgccgtgttccagtatgaag and 5′-cgatcatcagggctcgtacag.

Triplicate M. tuberculosis cultures were grown in modified 7H9 medium to an OD₆₀₀ of 1 and RNA was isolated as previously described using a chloroform-methanol and Trizol (Invitrogen) extraction (Perkowski et al., 2016 (link); Feltcher et al., 2015 (link)). RNA samples were treated with DNase (Promega, Madison, WI) and then column purified (Zymo RNA clean and concentrator Kit, Irvine, CA). Following RNA isolation, cDNA was synthesized with random primers using the iScript cDNA Synthesis Kit (Bio-Rad). Real-time PCR was completed using 25 ng of cDNA template in triplicate technical replicates using the SensiMix SYBR and fluorescein kit (Bioline, Toronto, Canada). Transcripts were normalized to the housekeeping gene sigA. Primer sequences are provided in the Key Resources Table.

Total RNA from cells and liver tissues was isolated using GenElute Total RNA Miniprep Kit (Sigma) and SV total RNA isolation system (Promega Corporation, Madison, WI, USA), respectively, according to the manufacturer's instructions. The RNA concentration was quantified with a UV spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). Total RNA (1 μg) was reverse transcribed using the iScript cDNA Synthesis Kit (Bio‐Rad, Hercules, CA, USA). All primers were purchased from Sigma‐Genosys (Haverhill, UK). Real‐time PCR was performed using 2× SensiMix SYBR and Fluorescein Kit (Bioline, QT615‐05, Luckenwalde, Germany), 20 ng cDNA and pretested gene‐specific primer sets (listed in Appendix Table S3). The cycling conditions for the Bio‐Rad CFX384 Real‐Time PCR detection system was 95°C for 10 min, 40 cycles of 95°C/15 s, 72°C/15 s, and 58°C/15 s. Finally, cycle threshold (Ct) values were normalized to reference gene GAPDH or 18S and fold changes in expression were calculated using the 2^−ΔΔCt method.

Total RNA from cells and liver tissues was isolated using GenElute Total RNA Miniprep Kit (Sigma) and SV total RNA isolation system (Promega Corporation) respectively according to manufacturer's instructions. The RNA concentration was quantitated by a UV spectrophotometer (NanoDrop Technologies). Total RNA (1 μg) was reverse transcribed using iScript cDNA Synthesis Kit (Bio‐Rad). All the primers were purchased from Sigma‐Genosys. Real‐time PCR was performed using 2× SensiMix SYBR and Fluorescein Kit (Bioline, QT615‐05), 20 ng cDNA and pre‐tested gene‐specific primer sets (Tables S3 and S4). The cycling conditions for the BioRad CFX384 Real‐Time PCR detection system were 95°C for 10 minutes, 40 cycles of 95°C/15 sec, 58°C/15 sec, and 72°C/15 sec. Finally, cycle threshold (Ct) values were normalized to reference gene GAPDH and fold changes in expression were calculated using the 2^−ΔΔCt method.

Expression of calmodulin- and calcineurin-encoding genes, cmdA and cnaA, respectively, was performed in the A. fumigatus (AF293) strain in the absence and presence of caspofungin. Conidia (10⁶/ml) were cultured in GMM broth under shaking conditions (200 rpm) for 20 h at 37°C. After 20 h, caspofungin (1 and 4 μg/ml) was added to the medium and cultures were incubated at 200 rpm for 4 h at 37°C. The resulting hyphae were harvested by vacuum filtration, washed extensively with cold sterile distilled water, and immediately frozen in liquid nitrogen. Total RNA was extracted using the RNeasy minikit (Qiagen) and treated with DNase I (Ambion). Total RNA (600 ng) was subjected to first-strand cDNA synthesis using the Tetro cDNA synthesis kit (Bioline). Real-time PCR assays were performed in triplicate using the iQ5 real-time PCR detection system (Bio-Rad) with 20-μl reaction volumes containing 2× Sensimix SYBR and fluorescein kit (Bioline), 0.2 μl of each primer, and 2 μl of a 1:5 dilution of the cDNA. The threshold cycle (2^−ΔΔCT) analytic method (11 (link)) normalized to beta-tubulin was used to calculate expression changes. Results are the means (± standard deviations) of results from three triplicate assays.