Genes were selected on the basis of their significance, potential interest or the pathway in which they are involved. One microgram of RNA was retrotranscribed using the High Capacity RNA-to-cDNA kit (Applied Biosystems). RT-qPCR reactions were carried out in triplicate using TaqMan® Gene Expression Assays (Supplementary Material, Table S7 ) on an ABI-Prism7500 Fast Instrument (Applied Biosystems). Gene expression was normalized to hydroxymethylbilane synthase gene (HMBS) (3 (link)), while microarray to HMBS and X-prolyl aminopeptidase (aminopeptidase P) 1, soluble gene (XPNPEP1) (63 (link)). Gene expression fold changes were calculated using the ΔΔCt method (64 (link)).
Abi prism7500 fast instrument
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI-Prism7500 Fast Instrument is a laboratory equipment designed for quantitative real-time PCR analysis. It is capable of performing fast thermal cycling protocols and provides precise data collection and analysis.
Automatically generated - may contain errors
2 protocols using abi prism7500 fast instrument
1
RT-qPCR Quantification of Gene Expression
2
Gene Expression Analysis in Colonic Tissue
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Total RNA extraction, cDNA synthesis, and gene expression analyses were performed. RNA extraction (RNA extraction mini kit, Qiagen, Hilden, Germany) from the colonic tissue was performed according to the manufacturer′s protocol. The primers and FAM-labeled probe sequences of TNF-α, IL-1β, IL-6, and β-actin used in this study are listed in Table 2 .
Real-time PCR amplification was performed using the Maxima Probe qPCR master mix (Thermo Fisher Scientific Inc., Waltham, MA, USA) with an ABI Prism 7500 fast instrument (Applied Biosystems, Foster city, CA, USA). Briefly, primers of target genes, probes, and cDNA samples were added into the Maxima Probe qPCR master mix. The reactions were performed by pretreatment with uracil-DNA glycosylase at 50 °C for 2 min and an initial preincubation at 95 °C for 10 min, followed by 40 amplification cycles, as follows: 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The gene expression levels of the genes of interest were normalized to β-actin, and the results were analyzed according to the ΔΔCt method. We reported the copy number change of the therapy group compared with that of the control group.
Real-time PCR amplification was performed using the Maxima Probe qPCR master mix (Thermo Fisher Scientific Inc., Waltham, MA, USA) with an ABI Prism 7500 fast instrument (Applied Biosystems, Foster city, CA, USA). Briefly, primers of target genes, probes, and cDNA samples were added into the Maxima Probe qPCR master mix. The reactions were performed by pretreatment with uracil-DNA glycosylase at 50 °C for 2 min and an initial preincubation at 95 °C for 10 min, followed by 40 amplification cycles, as follows: 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The gene expression levels of the genes of interest were normalized to β-actin, and the results were analyzed according to the ΔΔCt method. We reported the copy number change of the therapy group compared with that of the control group.
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