Colons were harvested from the TgM9 and WT littermate mice with and without CAC. These colons were then gently flushed with phosophate buffered saline (PBS) to remove feces. Total RNA was extracted from colonic tissues using the RNeasy mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Yield and quality of RNA were verified with a Synergy 2 plate reader (BioTek, Winooski, VT). Complementary DNA was generated from the earlier-described total RNA isolated using the Maxima first-strand complementary DNA synthesis kit (Thermo Scientific, Lafayette, CO). mRNA expression was quantified by quantitative real-time reverse-transcription polymerase chain reaction using Maxima SYBR green/ROX (6-carboxyl-X-rhodamine) quantitative polymerase chain reaction Master Mix (Thermo Scientific) and the following sense and antisense primers: Firmicutes, Bacteroidetes, A. muciniphila, REG3-a, REG3-b, REG3-g, S100A8, IL-22, IL-6, IL-1β, TNF-α, and IFN-γ (Supplementary Table 1 ).
Maxima sybr green rox 6 carboxyl x rhodamine quantitative polymerase chain reaction master mix
Manufactured by Thermo Fisher Scientific
Maxima SYBR green/ROX quantitative polymerase chain reaction Master Mix is a ready-to-use solution containing all the necessary components for real-time quantitative PCR, including a DNA polymerase, dNTPs, buffer, and SYBR Green I/ROX dye.
Automatically generated - may contain errors
2 protocols using maxima sybr green rox 6 carboxyl x rhodamine quantitative polymerase chain reaction master mix
1
Quantification of Gut Microbiome and Immune Markers
2
Colon Tissue RNA Extraction and qRT-PCR
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Total RNA was extracted from colonic tissues using the RNeasy mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Yield and quality of RNA were verified with a Synergy 2 plate reader (BioTek, Winooski, VT). Complementary DNA was generated from the earlier-described total RNA isolated using the Maxima first-strand complementary DNA synthesis kit (Thermo Scientific, Lafayette, CO). mRNA expression was quantified by quantitative real-time reverse-transcription polymerase chain reaction using Maxima SYBR green/ROX (6-carboxyl-X-rhodamine) quantitative polymerase chain reaction Master Mix (Thermo Scientific) and the following sense and antisense primers: IL6: 5’-ACAAGTCGGAGGCTTAATTACACAT-3’ and 5’-TTGCCATTGCACAACTCTTTTC-3’; CXCL2: 5’-CACTCTCAAGGGCGGTCAAA-3’ and 5'-TACGATCCAGGCTTCCCGGGT-3'; IL22: 5’-GTCAACCGCACCTTTATGCT-3’ and 5’-GTTGAGCACCTGCTTCATCA-3’; IL10: 5’-GGTTGCCAAGCCTTATCGGA-3’ and 5’-CTTCTCACCCAGGGAATTCA-3’; tumor necrosis factor α: 5’-AGGCTGCCCCGACTACGT-3’ and 5’-GACTTTCTCCTGGTATGAGATAGCAAA-3’; and 36B4: 5’-TCCAGGCTTTGGGCATCA-3’ and 5’-CTTTATCAGCTGCACATCACTCAGA-3’. Results were normalized by using the 36B4 housekeeping gene.
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