Total RNA was isolated from sorted cells with RNeasy kit (Qiagen). Reverse transcription was performed with the SuperScript III RT-PCR system (Invitrogen) and quantitative real-time reverse transcription (RT)-PCR with Taqman® Fast Universal PCR master mix, internal house keeping gene mouse (GAPD VIC-MGB dye) and specific target gene primers (FAM Dye) (Applied Biosystems) on Step-One- Plus machine. Relative expression was normalized to GAPD for Notch1-4 receptor and calculated as fold change compared to wild-type CD4+GFP− conventional T cells for Pofut1, Notch1, RBPJ and Rictor regarding the regulatory T cell-specific deficiency and fold change normalized to wild-type Treg cells for IFNg and IL12rb2.
Gapd vic mgb dye
Manufactured by Thermo Fisher Scientific
The GAPD VIC-MGB dye is a fluorescent label used in real-time PCR applications. It is designed to detect the presence of the GAPDH gene, which is a commonly used endogenous control in gene expression studies. The dye provides a reliable and sensitive method for quantifying GAPDH expression levels in a variety of sample types.
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2 protocols using gapd vic mgb dye
1
Regulatory T Cell Gene Expression
2
Regulatory T Cell Gene Expression
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Total RNA was isolated from sorted cells with RNeasy kit (Qiagen). Reverse transcription was performed with the SuperScript III RT-PCR system (Invitrogen) and quantitative real-time reverse transcription (RT)-PCR with Taqman® Fast Universal PCR master mix, internal house keeping gene mouse (GAPD VIC-MGB dye) and specific target gene primers (FAM Dye) (Applied Biosystems) on Step-One- Plus machine. Relative expression was normalized to GAPD for Notch1-4 receptor and calculated as fold change compared to wild-type CD4+GFP− conventional T cells for Pofut1, Notch1, RBPJ and Rictor regarding the regulatory T cell-specific deficiency and fold change normalized to wild-type Treg cells for IFNg and IL12rb2.
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