Total RNA was extracted from tissue samples and cell lines using the TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.,) according to the manufacturer's instructions. For mRNA reverse transcription, cDNA was synthesized using the ReverTra AceH qPCR RT kit (Toyobo, Osaka, Japan) with 1 mg total RNA. RT-qPCR was performed via SYBR Green assay (Invitrogen; Thermo Fisher Scientific, Inc.) using the SYBRH Select Master Mix for CFX (Invitrogen; Thermo Fisher Scientific, Inc.) as described (21 (link)). GAPDH was used as the control. The reverse transcription primers and qPCR primers of COUP-TFII and GAPDH were purchased from Thermo Fisher Scientific, Inc. COUP-TFII relative expression was evaluated by using the 2−ΔΔCq method (22 (link)). All PCR assays were performed in triplicate.
Protocol detail
COUP-TFII Expression Analysis via RT-qPCR
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Aceh
Assays
Cdna
Cell lines
Coup tfii
Gapdh
Mrna
Primers
Reverse transcription
Sybr green
Tissue
Trizol
Corresponding Organization :
Other organizations : First Affiliated Hospital of Xiamen University
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Variable analysis
independent variables
- None explicitly mentioned
- COUP-TFII relative expression
- GAPDH (used as the control)
- Positive control: None mentioned
- Negative control: None mentioned
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