Quantitative RT-PCR Analysis of Gene Expression

Total RNA was prepared and used to synthesize cDNA using SuperScript II reverse transcriptase, as described (Subramanian et al, 2015 (link)). qRT-PCR was performed using gene-specific primers (Table S1) and QuantiFast SYBR Green PCR Kit in Rotor-Gene Q real-time PCR machine (QIAGEN) and ViiA7 real-time PCR system (Thermo Fisher Scientific) according to manufacturer’s instructions, and relative gene expression was normalized with Gapdh expression. 2−∆∆Ct was used to compare the transcriptional differences. Cycle threshold (Ct) values of various genes were plotted against Ct values of Gapdh to determine a ratio between different treatment groups.

Table S1 Primers used for qRT-PCR

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Xie M., Chia R.H., Li D., Teo F.X., Krueger C, & Sabapathy K. (2021). Functional interaction between macrophages and hepatocytes dictate the outcome of liver fibrosis. Life Science Alliance, 4(4), e202000803.

Publication 2021

QuantiFast SYBR Green PCR Kit Rotor-Gene Q real-time PCR machine ViiA7 real-time PCR system

Cdna Gapdh Gene expression Genes Primers Q gene Real time pcr Reverse transcriptase Sybr green Transcriptional

Corresponding Organization : Institute of Molecular and Cell Biology

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Variable analysis

independent variables

Different treatment groups

dependent variables

Relative gene expression normalized with Gapdh expression
Transcriptional differences between treatment groups

control variables

Gapdh expression

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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