Myiq single colour real time pcr detection system

Intestinal specimens suspended in RNA lysis buffer containing β-mercaptoethanol were homogenized using the TissueLyser (Qiagen, Hilden, Germany) for 1 minute/25 Hz and total RNA was isolated using spin columns based on manufacturer's instructions (Promega, Madison, WI, USA). RNA was reverse-transcribed to cDNA using iScriptTM cDNA Synthesis kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). The PCR reaction mixture, containing iQSYBR Green Supermix (Bio-Rad Laboratories Inc.) was prepared based on manufacturer’s instructions and qRT-PCR analysis was performed using the MyiQ single-colour real time PCR detection system (Bio-Rad Laboratories Inc.) with MyiQ System Software Version 1.0.410 (Bio-Rad Laboratories Inc.). Commercially manufactured gene specific primers (Eurogentec, Seraing, Belgium) were used after confirmation of specificity and efficiency tests by qRT-PCR with dilution series of pooled cDNA at a temperature gradient (55°C to 65°C) for primer-annealing and subsequent melting curve analysis (S2 Table). The mRNA quantity was calculated relative to the expression of β-Actin (ACTB) reference gene.

Human endometrial stromal cells were lysed with TRIzol reagent (Sigma, St. Louis, MO, USA) and total RNA was extracted in strict accordance with the instructions. Two micrograms of total RNA was added to a 20 μL system and reverse-transcribed into cDNA using 5 × All-In-One RT Master Mix (Abm, Canada). A SYBR Green PCR kit and MyiQ Single Colour Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) were employed for quantitative real-time PCR (qRT-PCR). The primer sequences used were as follows: KLF4, 5′-AGAGTTCCCATCTCAAGGCA-3′ and 5′-GTCAGTTCATCTGAGCGGG-3′; ATG5, 5′-AAAGATGTGCTTCGAGATGTGT-3′ and 5′-CACTTTGTCAGTTACCAACGTCA-3′; dPRL, 5′-CACTACATCCATAACCTCTC-3′ and 5′-ATGCTGACTATCAAGCTCAG-3′; IGFBP-1, 5′-TATGATGGCTCGAAGGCTCTC-3′ and 5′-GTAGACGCACCAGCAGAGTC-3′ and 18S rRNA, 5′-CGGCTACCACATCCAAGGAA-3′ and 5′-CTGGAATTACCGCGGCT-3′. The fold changes in the expression of each gene were measured by the 2^−ΔΔCT method. The internal reference gene was 18S rRNA.

Levels of mRNA were measured exactly as described previously^{38 (link)}. Briefly, interscapular BAT was isolated in Invitrogen TRI Reagent, lysed and homogenized with QuickPrep Adaptor (Fisher Scientific). RNA isolation was performed according to the manufacturer (Fisher Scientific) and cDNA synthesized using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Fisher Scientific), Invitrogen RNaseOUT Recombinant Ribonuclease Inhibitor (Fisher Scientific) and T100 Thermal cycler (Bio-Rad). Gene expression of cDNA (30 ng) was determined using the qPCRBIO Probe Mix No-ROX (PCR Biosystems), performed on the MyiQ Single-Colour Real-Time PCR Detection System (Bio-Rad). Samples were measured in duplicates and the 2^−∆∆Ct method employed to determine fold change in gene expression. The target genes were normalized to the housekeeping gene, peptidylprolyl isomerase A (PPIA). All Applied Biosystems TaqMan Gene Expression Assay (FAM-MGB) (Major Resources Table) were purchased from Fisher Scientific.