Total RNA was extracted with RIPA lysis buffer (Beyotime Institute of Biotechnology). cDNA was then reverse-transcribed according to the methods described in a previous study. The mRNA expression was measured via qRT-PCR (SYBR Green method).39 The SYBR Green reagent was obtained from Solarbio Life Sciences (Beijing, China). The relative mRNA levels were normalized to GAPDH and calculated using the 2−ΔΔCt method as described in a previous study.40 (link) The following primers were used in the present study: ROCK-1 (Forward: 5′-ACCTGTAACCCAAGGAGATGTG-3′, Reverse 5′-CACAATTGGCAGGAAAGTGG-3′), and Rac1 (Forward 5′-ATGCAGGCCATCAAGTGTGTGG-3′, Reverse: 5′-TTACAACAGCAGGCATTTTCTC-3′). The experiments were performed in triplicate and repeated three times with similar results.
Protocol detail
Quantifying ROCK-1 and Rac1 mRNA Levels
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Buffer
Cdna
Gapdh
Mrna
Primers
Ripa
Sybr green
Corresponding Organization :
Other organizations : Second Xiangya Hospital of Central South University, Central South University
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Variable analysis
independent variables
- ROCK-1 (Forward: 5′-ACCTGTAACCCAAGGAGATGTG-3′, Reverse 5′-CACAATTGGCAGGAAAGTGG-3′)
- Rac1 (Forward 5′-ATGCAGGCCATCAAGTGTGTGG-3′, Reverse: 5′-TTACAACAGCAGGCATTTTCTC-3′)
- MRNA expression
- GAPDH
- The relative mRNA levels were normalized to GAPDH and calculated using the 2^(-ΔΔCt) method as described in a previous study.
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