Quantitect reverse transcriptase kit

RNA (Qiagen) was isolated from 5-day-old seedlings harvested before or after NaCl (200 mM/6 h) treatment. Afterwards cDNA was synthesized (QuantiTect Reverse transcriptase kit, Qiagen) according to the manufacturer’s instructions. Primers (Supplementary Table S1) were designed using QuantPrime software discriminating splice variants and span exon–exon borders (http://quantprime.mpimp-golm.mpg.de/, last accessed 24 February 2017) (Arvidsson et al., 2008 (link)). The qPCR was performed with Applied Biosystems 7500 (Fast) and the Sequence Detection Software 1.3.1 from Applied Biosystems. Raw data analysis and calculation of the efficiency (E) and cycle threshold (CT) values were carried out with the PCR-Miner software http://ewindup.info/miner/, last accessed 24 February 2017 (Zhao and Fernald, 2005 (link)). The relative transcript abundance was calculated as (1+E)^−CT for every well and normalized against the geometric mean of the reference genes ubiquitin 5, tubulin 9, and ribosomal protein S16 (Scheler et al., 2015 (link); Supplementary Table S1).

For measurement of target gene expression, gonococci were harvested at mid- or late-log phase and the pellets were stored at −70 °C. RNA was purified by Trizol extraction as per manufacturer instructions (Thermo Fisher Scientific, Waltham, MA) followed by Turbo DNA-free (Ambion, Austin, TX) treatment. cDNA was generated using a QuantiTect reverse transcriptase kit (Qiagen, Venlo, Netherlands). We validated our qRT-PCR methods by examining primer efficiency, primer specificity (melt temperature) and linear dynamic range for each primer pair utilized herein. For additional information about our validation results, see Supplemental Fig. S6. For qRT-PCR analysis, the normalized expression of each target gene was calculated using 16 S rRNA as a housekeeping reference gene^{33 (link)}. As an additional internal control, significance was confirmed using recA as the reference gene (data not shown). All qRT-PCRs were performed in technical and biological triplicates.

Total RNA was extracted using the RNeasy Lipid Tissue Mini kit and Qiacube (Qiagen, Valencia, CA) from flash-frozen hind leg biceps femoris skeletal muscle or subcutaneous adipose tissue. cDNA was synthesized using the Quantitect Reverse Transcriptase kit (Qiagen, Valencia, CA) and then used to measure expression of GLUT4, HIF1α, FGF21, PPARγ, MAP1LC3A, and BECN1 by qPCR (ABI Prism 7500 PCR System, Applied Biosystems, Foster City, CA). FastStart Universal Probe Master (Rox) mix assay reagents were purchased from Roche. Primers were purchased from Integrated DNA Technology (IDT) (Coralville, IA). The endogenous control (18S rRNA) was purchased from Applied Biosystems. RT-PCR analysis for TRPC1 transcripts was done with primers from the eighth and ninth exons (forward, 5′-GCAACCTTTGCCCTCAAAGTG and reverse, 5′-GGAGGAACATTCCCAGAAATTTCC) after the EcoRI site (Eurofins MWG Operon, Huntsville, AL).

MM6 cells (1.5 × 10⁶) were exposed to one of the short-chain alcohols (ethanol, propanol or isopropanol) at a 50-mM concentration or 1 μM dexamethasone (Dex) for 24 h in the presence or absence of 5 μM mifepristone. The cells were harvested and washed twice with 1x PBS. Total RNAs were extracted using the Qiagen RNeasy Kit. The cDNAs were synthesized using the Quantitect Reverse Transcriptase Kit (Qiagen). Human GILZ primers (GILZ-F: 5′-CATGGAGGTGGCGGTCTATC-3′ and GILZ-R: 5′-CACCTCCTCTCTCACAGCGT-3′) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers (GAPDH-F: 5′-AAGGTCGGAGTCAACGGATTTGGT-3′ and GAPDH-R: 5′- ACAAAGTGGTCGTTGAGGGCAATG-3′) were used at a final concentration of 500 nM, as published previously (1 (link)). The final reaction for each sample was brought to a total volume of 20 μl with RT SYBR green qPCR mastermix (Qiagen). All reactions were carried out in duplicate on a CFX96 system (Bio-Rad Laboratories, Hercules, CA) for quantitative real-time PCR (qPCR) detection. The qPCR data were analyzed by the comparative Ct (ΔΔCT) method. The expression of GILZ of each treated group was compared to that of GAPDH, and normalized to the non-treatment group.

Pdgfc expression was assessed in primary lung fibroblasts before immortalization. Lungs or tumors were homogenized in lysis buffer using a Precellys tissue homogenizer. RNA was isolated using Qiagen RNeasy Plus Micro (homogenized tissues) or RNeasy Plus Mini (cells) kits. cDNA was generated using the QuantiTect reverse transcriptase kit (Qiagen) or the SuperScript IV kit (Invitrogen) with random hexamers for overexpression lines. RT–qPCR was performed with Taqman Gene Expression Assay probes (Supplementary Table 3) on a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, 1.7.1). Each reaction was performed in triplicate, and relative expression levels were normalized to B2m or B2M and/or Ipo8 or IPO8. When calculating the relative expression across independent cell lines, multiple house-keeping genes were used (B2m, Ipo8, Ubc and 18s).