Total RNA was extracted from the collected cells or colonic tissues using TRIzol reagent (Life Technologies; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions and reverse transcribed into complementary DNA using Prime Script RT Master Mix (Takara Biotechnology, Inc.). qPCR was performed using the LightCycler® 480 SYBR I Master Mix (Roche Diagnostics), running on a Roche LightCycle R480 system (Roche Diagnostics). Gene expression was normalized relative to GAPDH and calculated using the 2−ΔΔCq method (26 (link)). The primer sequences are presented in Table I .
Lightcycler 480 sybr 1 master mix
Manufactured by Roche
Sourced in Switzerland
The LightCycler® 480 SYBR I Master Mix is a ready-to-use reaction mix for real-time PCR amplification using SYBR® Green I dye. It is designed for reliable and sensitive detection of target sequences.
Automatically generated - may contain errors
4 protocols using lightcycler 480 sybr 1 master mix
1
RNA Extraction and qPCR Analysis
2
Quantification of Gene Expression in Colon Tissues
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RNA was extracted from colon tissue or cells using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. The reverse transcription (cDNA) was synthesized from 1 μg of total RNA with Prime Script RT Master Mix (Takara Biotechnology, Dalian, China). Quantitative Real-time PCR was performed using 1 μl first-strand cDNA with the LightCycler® 480 SYBR I Master Mix (Roche, Switzerland), in a final volume of 10 μl. All samples were run in triplicate and underwent 45 amplification cycles in a Roche LightCycler R480 (Roche, Switzerland). The relative fold change of mRNA expression was measured by using 2−ΔCT method and normalized. Primers used can be seen on Additional file 1 : Table S1.
3
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted from tissues or cells using TRIzol reagent (Invitrogen, 15596018) according to the manufacturer’s instructions and reversed to cDNA using Prime Script RT Master Mix (Takara Biotechnology, RR036). qPCR was performed using the LightCycler® 480 SYBR I Master Mix (Roche Diagnostics), running on a Roche LightCycle R480 system (Roche Diagnostics). The relative fold change of mRNA expression was normalized relative to GAPDH and measured by the 2−ΔCT method. The primer sequences are presented in Table 1
4
Quantitative Gene Expression Analysis
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RNA was extracted from colon tissue or cells using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Reverse transcription (cDNA) was synthesized from 1 μg of total RNA with Prime Script RT Master Mix (Takara Biotechnology, Dalian, China). Quantitative Real-time PCR (qPCR) was performed using 1 μl of first-strand cDNA with the LightCycler® 480 SYBR I Master Mix (Roche, Switzerland) at a final volume of 10 μl. All samples were run in triplicate and underwent 45 amplification cycles on a Roche LightCycler R480 (Roche, Switzerland). The relative fold-change in mRNA expression was measured by using the 2−ΔCT method and normalized. The primers used are listed in Additional file 1 : Table S1.
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