Cfx96 connect real time pcr system instrument

Total RNA was extracted from the spinal cord using Trizol (Ambion). Two micrograms of total RNA were used in first-strand cDNA synthesis (Promega, Madison, WI) according to the manufacturer’s instructions. PCRs were performed with a Bio-Rad CFX96 Connect Realtime PCR System instrument and software (Bio-Rad Laboratories). The PCR conditions were 15 min at 95°C followed by 40 cycles of two-step PCR denaturation at 94°C for 15 s, annealing extension at 55°C for 30 s and extension at 72°C for 30 s. Each sample contained 500 ng cDNA in 2X QuantiTech SYBRGreen PCR Master Mix and gene-specific primers for Tnfa, Il1b, and Il6 were purchased from Qiagen (Hilden, Germany). The relative expression of mRNA was normalized to Gapdh as housekeeping gene, and the data were analyzed according to the 2–ΔΔCT method.

Total RNA, isolated from the liver (n = at least 8 animals each group), was obtained by the extraction using TRIzol Reagent (Bio-Rad Laboratories) and following a specific RNA extraction kit (NucleoSpin^®, MACHEREY-NAGEL GmbH & Co, Düren, Germany), according to the manufacturer’s instructions. cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA) as previously described [31 (link)], from 8 µg total RNA. PCRs were performed with a Bio-Rad CFX96 Connect Real-time PCR System instrument and software (Bio-Rad Laboratories, Segrate, Milan, Italy). The PCR conditions were previously reported [24 (link)]. Each sample contained 500 ng cDNA in 2X QuantiTech SYBRGreen PCR Master Mix and primers pairs to amplify G6pc, Pck1, Fasn, Srebf1, Pparg, Tnfa, Ifng, Il6, Tgfb, Col1a1, Col3a1, Myd88, NACHT, LRR and PYD domain-containing protein (Nlrp3), Pycard, Casp1, Il1b, Nfkb1, Ccl2, and Itgax (Qiagen, Hilden, Germany). The relative amount of each studied mRNA was normalized to Actb as a housekeeping gene, and the data were analyzed according to the 2^−ΔΔCt method.

Real-time PCR was performed using a CFX96 Connect Real-time PCR System instrument and an iQ™ SYBR^® Green Supermix in sealed 96-well microplates, from Bio-Rad Laboratories (Hercules, CA, USA). PCR reaction mixtures were composed of 1 μL of DNA (equilibrated to 20 ng), 1 μL of each primer (final concentration of 0.1 μM), 5 μL of iQ™ SYBR^® Green and 2 μL of water. The conditions for PCR amplifications and the primer sequences (STAB VIDA, Lisboa, Portugal) used to target the 16S rRNA gene of the bacteria are listed in Table 1.
Data were processed and analyzed using the CFX Maestro software (Bio-Rad Laboratories (Hercules, CA, USA) and standard curves were constructed using serial tenfold dilutions of bacterial genomic DNA, according to the following webpage http://cels.uri.edu/gsc/cndna.html (accessed on 20 May 2023). Bacterial genomic DNA used as a standard (Table 1) was obtained from DSMZ (Braunschweig, Germany). The copy number of the 16S rRNA gene for each bacterial strain used as a standard and genome size was obtained from the NCBI Genome database (www.ncbi.nlm.nih.gov (accessed on 20 May 2023)). Data are presented as the mean values of the duplicate PCR analysis. The Firmicutes/Bacteroidetes ratio was obtained by dividing the number of copies of the Firmicutes division by the number of copies of Bacteroidetes division.