Cfx connect real time pcr cycler

mRNA expression of vastus lateralis muscle was conducted as follows (Reidy et al. 2018a (link)). Total RNA was isolated by homogenizing 10–15 mg tissue with a hand-held homogenizer in a solution containing Tri reagent LS (Molecular Research Centre, Cincinnati, OH, USA). The RNA was separated and precipitated using chloroform and isopropanol. Extracted RNA was washed with ethanol then suspended in nuclease-free water with EDTA. RNA concentration was determined using the EPOCH (Take3; BioTek) spectrophotometer. cDNA was synthesized using a commercially available kit (iScript, BioRad, Hercules, CA, USA). All isolated RNA and cDNA samples were stored at −80°C until analysis. Real-time PCR was carried out with a CFX Connect real-time PCR cycler (BioRad) under similar protocol as reported previously (Drummond et al. 2014 (link); Reidy PT 2016) using SYBR green custom designed primers for beta 2-microglobulin (β2M), collagen type I alpha 1 chain (Col1a), collagen type III alpha 1 chain (Col3a), Fibroblast growth factor 21 (FGF21), cluster of differentiation 68 (CD68), and C-C motif chemokine ligand 2 (CCL2) which have been previously described (Kwon et al. 2015 (link)). Cycle threshold values of target genes were normalized to β2M then fold change values were calculated (ΔΔCt). β2M remained stable across the interventions.

qRT-PCR was used to determine the expression levels of dipA (PA5017) using 1 µg of total RNA isolated from wild type P. aeruginosa PAO1, PAO1/pJN-bdlA, and PAO1/pJN-bdlA-G31A grown planktonically. Isolation of mRNA and cDNA synthesis was carried out as previously described (Petrova & Sauer, 2010 (link), Petrova & Sauer, 2011 (link)). qRT-PCR was performed using the CFX Connect realtime PCR cycler (Biorad) and the SsoAdvanced SYBR Green supermix (Biorad), with oligonucleotides listed in Table S3. mreB was used as control. The stability of mreB levels were verified by 16S RNA abundance using primers HDA1/HDA2 (McBain et al., 2003 (link)). Relative transcript quantitation was accomplished using the CFX Connect software (Biorad) by first normalizing transcript abundance (based on Ct value) to mreB followed by determining transcript abundance ratios. Melting curve analyses were employed to verify specific single product amplification.

RNA and cDNA samples were analyzed on an Agilent 2100 bioanalyzer using the 6000 Nano or Pico Kit, respectively, according to manufacturer's instructions (Agilent Technologies, Waldbronn, Germany). 1.2 μg of total RNA was converted to cDNA using the SuperScript III First-Strand Synthesis System with poly-dT primer extension and final RNase H treatment (Life Technologies). For detecting CYC1 transcripts, cDNA samples were subjected to PCR using the following primers: 5′-region: Forward (FW): 5′-GTCGTCGAAGTCTGGCCTTT-3′, Reverse (RV): 5′-CACGGTGAGACCACGGATAG-3′; Central-region: FW: 5′-GCCTCCTCTCTTCCTTGGAC-3′, RV: 5′-TCTTCATTGGGGCCGTCTTG-3′; 3′-region: FW: 5′-GGCATGGTGGTGAGGACTAC-3′, RV: 5′-CCCATGCGTTTTCGATGGTC-3′. For each sample, 1 μl of the cDNA solution (57 ng total RNA equivalent) was serially diluted 1:10, 1:100, 1:1,000, and 1:10,000, and PCR amplified in a Bio-Rad CFX Connect Real-Time PCR cycler using 0.2 μM primers and the iTaq Universal SYBR Green reagent (Bio-Rad, Hercules, CA).