Total RNA was extractr’ed from organoids in TRIZOL according to manufactures instructions, with the addition of glycogen. cDNA was generated from 500 ng RNA via the Verso cDNA synthesis kit (ThermoFisher #AB-1453). gDNA was isolated from 1 ml aliquots of stool-based bioreactors using the Zymo Quick-DNA Fecal/Soil Microbe Kits according to the manufacturer’s instructions. Quantitative real time PCR (qPCR) was performed using a Bio-Rad CFX96 Real Time qPCR machine (Bio-Rad). Forward and reverse primers were added to SYBR Green mastermix (Genesee Scientific #17-501DP) and cDNA. Epithelial genes were normalized to the housekeeping gene 18S and relative expression was calculated using the ddCT method. Bacterial colony forming units (CFUs) were calculated from CT values based on standard curves as previously described (Engevik et al., 2013 (link)).
Cfx96 real time qpcr machine
Manufactured by Bio-Rad
Sourced in United States
The CFX96 Real Time qPCR machine is a compact, high-performance real-time PCR system designed for precise and reliable nucleic acid quantification. It features a 96-well format, supports multiple fluorescent dyes, and enables real-time monitoring of amplification during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Verso cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Quick dna fecal soil microbe kit, by Zymo Research (1 mentions)
Itaq universal sybr green supermix, by Bio-Rad (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
3 protocols using cfx96 real time qpcr machine
1
Quantitative Analysis of Organoid and Gut Microbiome
2
Validating RNAseq Data by qRT-PCR
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To confirm the RNAseq results, quantitative real-time PCR (qRT-PCR) was carried out using a CFX96 RealTime qPCR machine (Bio-Rad). The SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific) was used to produce cDNA from 1 μg of the isolated RNA following the manufacturer’s instructions. qRT-PCR was performed using the primers listed in the supplementary data using iTaq Universal SYBR Green Supermix (Bio-Rad). Each reaction was carried out in triplicate and elongation factor 1 alpha (ef1α, zebrafish) or GAPDH (human cells) was selected as an internal control to normalize the RNA levels. The 2−ΔΔCt method was used to quantify gene expression. Three biological replicates for each mutant and for the controls (3 batches of 30 pooled embryos each) were used for the RNA analysis.
3
Quantifying Inflammatory Chemokine Expression
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Synovium and hyaline cartilage samples were weighed and ground in liquid nitrogen. Trizol reagent (Invitrogen Life Technologies, USA) was added immediately to the ground tissue (1 ml/ 100 mg tissue), and samples were incubated at room temperature for 10 min. Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer's instructions. The quality of the RNA samples was quantified using a NanoDrop spectrophotometer (NanoDrop Technologies, USA). A QuantiTect Reverse Transcription Kit (Qiagen, Germany) was used for the reverse transcription of mRNA. qRT-PCR of target mRNAs was performed using a CFX96 real-time qPCR machine (Bio-Rad, USA) using the QuantiTect SYBR Green PCR Kit (Qiagen, Germany). The specific primers used to amplify human CCL2, CCL3 and CCL4 were as follows: CCL2, sense-AGAATCACCAGCAGCAAGTGTCC and antisense-TCCTGAACCCACTTCTGCTTGG; CCL3, sense-ACTTTGA-GACGAGCAGCCAGTG and antisense-TTTCTGGACCCACTCCTCACTG; and CCL4, sense-GCTTCCTCGCAACTTTGTGGTAG and antisense-GGTCATACACGTACTCCTGGAC. Each value was normalized to the expression of GAPDH. The relative expression of genes and mRNAs was calculated using the comparative CT method (DDCt).
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