Total RNA was extracted using the TRIzol reagent (Ambion, Life Technologies, USA), and RNA quality was subsequently assessed using the Infinite M200 PRO NanoQuant absorbance microplate reader (TECAN, Chapel Hill, NC, USA). Reverse transcription was conducted with a SuperRT cDNA Synthesis Kit (CWbio, Beijing, China) and real-time quantitative reverse transcriptase-polymerase chain reactions (qRT-PCR) were performed using the Ultra SYBR Mixture with low ROX (CWbio), as previously described (29 (link)). Gene expression was calculated using the 2−ΔΔCt method and Actb expression was used as the internal control. PCR primer sequences used in qPCRs are listed in Supplementary Table 1
Ultra sybr mixture with low rox
Manufactured by CWBIO
Sourced in China, United States
Ultra SYBR Mixture with low ROX is a qPCR reagent designed for sensitive and accurate real-time PCR amplification. It contains a proprietary SYBR Green I dye and low concentration of ROX reference dye for normalization of fluorescent signals.
Automatically generated - may contain errors
2 protocols using ultra sybr mixture with low rox
1
Quantitative Gene Expression Analysis
2
Gene Expression Analysis by qRT-PCR
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Total RNA was isolated using TRIzol extraction (Invitrogen, Carlsbad, CA, USA), and RNA quality was assessed using the Infinite M200 PRO NanoQuant absorbance microplate reader (TECAN, Chapel Hill, NC, USA). One microgram (1 μg) of RNA was reverse transcribed with SuperRT cDNA Synthesis Kit (CWbio, Beijing, China) according to the manufacturer's instructions. All real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) reactions were performed with Bio-Rad thermocycler (Bio-Rad, Hercules, CA, USA) using Ultra SYBR Mixture with low ROX (CWbio). Gene expression data were normalized to the internal control of Gapdh expression using the delta-delta comparative threshold cycle (2 ÀΔΔCt ) method. PCR primer sequences are listed in Appendix S1, Table S2. This experiment was performed in triplicate.
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