Cfx connect rt pcr system

RNA isolated from cells from biological triplicate experiments using TRIzol RNA Isolation Reagents (Thermo Fisher Scientific, Waltham, MA, USA) was measured using a NanoDrop™ Lite Spectrophotometer (Thermo Fisher). RNA (500 ng) from each sample was reverse-transcribed using the SuperScript^® IV First-Strand Synthesis System (Thermo Fisher). c-Myc, and GAPDH were amplified in duplicate using Power UP SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) in the CFX Connect RT-PCR system (Bio-Rad, Hercules, CA, USA), using previously published primers [48 (link)]. The ∆Ct method for relative quantification of gene expression was used to determine mRNA expression levels [48 (link)]. c-Myc mRNA levels normalized to GAPDH mRNA levels at serial time points were compared to time 0 (pre-treatment) levels, defined as 1.

The total RNA was isolated from mature SGBS adipocytes using the RNeasy^® Mini Kit (Qiagen, Hilden, Germany), including DNase digestion (RNase-Free DNase Set, Qiagen, Hilden, Germany). Gene expression was quantified by reverse transcription of 300 ng RNA (QuantiTect Reverse Transcription Kit from Qiagen, Hilden, Germany) and subsequent real-time PCR (RT-PCR) (iTaq Universal SYBR Green Supermix, CFX Connect RT-PCR system; Bio-Rad, Munich, Germany) of the corresponding cDNA. The following primer sequences (forward/reverse) were used:

Human CAMP:

5′-TAGATGGCATCAACCAGCGG-3′/5′-CTGGGTCCCCATCCATCGT-3′

Human DPP4:

5′-TTCTGCTGAACAAAGGCAATGA-3′/5′-CTGTTCTCCAAGAAAACTGAGCTG-3′

Human FABP4:

5′-ATGGGGGTGTCCTGGTACAT-3′/5′-CTTTCATGACGCATTCCACCA-3′

Human GAPDH:

5′-GAGTCCACTGGCGTCTTCAC-3′/5′-CCAGGGGTGCTAAGCAGTT-3′

Expression levels of the target gene were normalized to the gene expression of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All oligonucleotides used were purchased from Metabion, Martinsried, Germany.

Total RNA extraction was done using TRIzol reagent (Sigma, #T9424). DNase I-treated (Thermo Scientific, #EN0521) RNA was used to synthesize first-strand complementary DNA (cDNA). cDNA was synthesized using an iScript cDNA synthesis kit (Bio-Rad, #1708891) with oligodT, as per the manufacturer’s protocol. Templates were amplified using gene-specific primers for PCNA (forward: 5′-TCACAGGGCAGTGTCTTCATT-3′; reverse: 5′-GGGTGACTGTAGCTGGGAAT-3′), Ki67 (forward: 5′-GACAGTACCGCAGATGACTC-3′; reverse: 5′-TACGTCCAGCATGTTCTGAGG-3′), vimentin (forward: 5′-TCTACGAGGAGGAGATGCGG-3′; reverse: 5′-GGTCAAGACGTGCCAGAGAC-3′), N-Cad (forward: 5′-CGAATGGATGAAAGACCCATCC-3′; reverse: 5′-GGAGCCACTGCCTTCATAGTCAA-3′), and IL-6R (forward: 5′-GGATGGTCAAGGACCTCCAG-3′; reverse: 5′-CTGGATTCTGTCCAAGGCGT-3′), taking β-actin (forward: 5′-CCACCATGTACCCTGGCATT-3′; reverse: 5′-CGGACTCGTCATACTCCTGC-3′) as housekeeping control, and detected using SYBR Green Supermix (Bio-Rad, #170-8882AP) in a CFX Connect RT-PCR system (Bio-Rad). The relative mRNA expression was calculated by Pfaffl’s method (43 (link)). The melting temperature for the PCRs was 56.4°C for PCNA, 56.7°C for Ki67, 60.4°C for vimentin, 61.4°C for N-Cad, 64.1°C for IL-6R, and 61.7°C for β-actin.

Total RNA was isolated using TRIzol reagent (Invitrogen); complementary DNA (cDNA) was synthesized using GeneSure First Strand cDNA Synthesis kit (Genetix) with oligodT following manufacturer's instructions. cDNA templates were amplified for ABCB1 gene in CFX Connect RT-PCR System (Bio-Rad) and detected using SYBR Green (Bio-Rad). As a control, GAPDH was amplified. The primer sequence for ABCB1 was forward -5’- GGGATG GTCAGTGTTGATGGA -3’ and reverse 5’-GCTATCGTGGTGGCAAACAATA-3’ and for GAPDH was forward- 5’-TGATGACATCAAGAAGGTGGTGAAG-3’ and reverse- 5’-TCC TTGGAGGCCATGTGGGCCAT-3’. The relative RNA expression was calculated using the Livak method [19 (link)].

Total RNA was extracted from cells using Isogen II (Nippon Gene, Tokyo, Japan), following the manufacturer's protocol. Purified RNA (0.5 μg) was subjected to reverse transcription with Revertra Ace (TOYOBO, Osaka, Japan). Quantitative RT‐PCR was performed using a CFX Connect™ RT‐PCR System (Bio‐Rad, Hercules, CA, USA) with THUNDERBIRD SYBR qPCR Mix (TOYOBO). Quantitative PCR data were processed using a standard curve method. Ribosomal protein S18 (Rps18) mRNA expression was used for normalization. The sequences of the primers used in this analysis are shown in Table 1.