Cfx connect real pcr system

The total RNA was extracted from cells with a Trizol reagent and the cDNAs were synthesized using the superscript III preamplification system. The PCR reaction was carried out in the Biometra T3000 thermocycler (Jena, Germany) and the PCR products were separated with 2% agarose gel electrophoresis. The real-time PCR (qPCR) was performed using a CFX Connect Real-PCR system (Bio-Rad Laboratories, Foster City, CA, USA). TaqMan™ gene expression master mix and polymerase chain reaction (PCR) FAM dye-labeled TaqMan MGB probes for human WISP1 (Hs04234730_m1 for total isoforms and Hs00180245 for WISP1v1), α-SMA (Hs00426835_g1), Tagln (Hs01038777_g1), IL-6 (Hs00985639_m1), GDF15 (Hs00171132_m1), COL1A2 (Hs01028956_m1), COL3A1 (Hs00943809), CXCL5 (Hs01099660_g1), NDRG1 (Hs00608387_m1), N-cadherin (Hs00169953_m1), snail (Hs00195591_m1), slug (Hs00161904_m1), vimentin (Hs00185584_m1), and β-actin (Hs01060665_g1) were purchased from Thermo Fisher Scientific Inc. (Vilnius, Lithuania). The mean cycle threshold (Ct) values were calculated for β-actin and the target genes using Bio-Rad CFX manager 3.1 (Bio-Rad Laboratories, Foster City, CA, USA). The Ct values for the target genes were normalized against the β-actin control probe to calculate the Δ Ct values as, described previously [55 (link)].

The cDNAs were synthesized using the superscript III pre-amplification system (Invitrogen) after the total RNA was extracted from the cells using a TRIzol reagent (ambion, Life technologies, Carlsbad, CA, USA). The ARv7 primers (5′-GGAAATGTTATGAAGCAGGGATG-3′ and 5′-GGTCATTTGAGATGCTTGCA), ARFL primers (5′-TGCAGCCTATTGCGAGAGA-3′ and 5′-TGATCTCTGCCATCATTTCC), and 18S (5′-ACCGCAGCTAGGAATAATGGA-3′ and 5′-GCCTCAGTTCCGAAAACCA-3′) were used according to the previous report [50 (link)]. The qPCR reactions were performed in a 12 μL reaction volume that consisted of 6 μL of 2 x iQ^TM SYBR Green supermix (Bio-Rad Laboratories, Foster City, CA, USA), 1 μL of the cDNA sample, 1 μL of gene-specific primers (5 μM), and 4 μL of H₂O. For the MALT1 and NDRG1 assays, TaqMan^TM gene expression master mix and FAM dye-labeled TaqMan MGB probes for human MALT1 (Hs01120052_m1), NDRG1 (Hs00608387_m1), and β-actin (Hs01060665_g1) from Thermo Fisher Scientific Inc. (Vilnius, Lithuania) were used as described previously [35 (link)]. The qPCR analysis was performed using a CFX Connect Real-PCR system (Bio-Rad Laboratories, Foster City, CA, USA). The cycle threshold (Ct) values for the target genes were normalized against the 18S or β-actin control probe to calculate the mean cycle threshold (ΔCt) values using the Bio-Rad CFX manager 3.1 (Bio-Rad Laboratories, Foster City, CA, USA).