Total RNA extraction was performed using TRI-zol reagent (15596026, Thermo Fisher Scientific, Inc., Waltham, MA, USA) [20 (link)], following the manufacturer’s instructions. Reverse transcription was carried out according to the Goldenstar™ RT6 cDNA Synthesis Kit Ver.2 (TSK302M, Tsingke Biotechnology Co., Ltd., Beijing, China) protocol. mRNA expression was assessed using the 2× T5 Fast RT-qPCR Mix (SYBR Green I) kit (TSE002, Tsingke Biotechnology Co., Ltd., Beijing, China) with the following reaction conditions: 95 °C for 30 seconds, 95 °C for 5 seconds, 60 °C for 30 seconds, for 40 cycles. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal reference gene, and the relative gene expression was calculated using the 2−ΔΔCt method. Specific primers for RT-qPCR are provided in Supplementary Table 3 .
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RNA Extraction and RT-qPCR Protocol
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Corresponding Organization :
Other organizations : Affiliated Hospital of Guizhou Medical University, Guizhou University
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Variable analysis
independent variables
- Total RNA extraction method (TRI-zol reagent)
- Reverse transcription kit (Goldenstar™ RT6 cDNA Synthesis Kit Ver.2)
- RT-qPCR kit (2× T5 Fast RT-qPCR Mix (SYBR Green I))
- MRNA expression
- Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal reference gene
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