Total RNA was isolated using RNeasy Plus Mini kit (QIAGEN) as per the manufacturer’s protocol, subsequently used to generate cDNA with LunaScriptTM RT SuperMix Kit (New England Biolabs). Realtime PCR was performed using SYBR green gene expression assays (New England Biolabs) on a QuantStudio 6 instrument (Applied Biosystems). Gene expressions were normalized to the endogenous GAPDH housekeeping gene. Please refer to Supplemental Data 2.
Sybr green gene expression assays
Manufactured by New England Biolabs
SYBR green gene expression assays are fluorescent dyes used to detect and quantify DNA amplification during real-time PCR. They bind to double-stranded DNA, emitting fluorescent signals that are proportional to the amount of DNA present in the sample.
Automatically generated - may contain errors
3 protocols using sybr green gene expression assays
1
RNA Isolation and qRT-PCR Analysis
2
RNA Isolation and Real-Time PCR Analysis
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Total RNA was isolated using RNeasy Plus Mini kit (Qiagen) as per the manufacturer’s protocol, subsequently used to generate cDNA with LunaScriptTM RT SuperMix Kit (New England Biolabs). Real‐time PCR was performed using SYBR green gene expression assays (New England Biolabs) on a QuantStudio 6 instrument (Applied Biosystems). Gene expressions were normalized to the endogenous GAPDH housekeeping gene.
3
Quantitative Gene Expression Analysis
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Total RNA was isolated using RNeasy Plus Mini kit (Qiagen, catalog no. 74134) as per the manufacturer's protocol, subsequently used to generate cDNA with LunaScript RT SuperMix Kit (New England Biolabs, catalog no. E3010S). Real-time PCR was performed using SYBR green gene expression assays (New England Biolabs, catalog no. M3003S) on a QuantStudio 6 instrument (Applied Biosystems). Gene expressions were normalized to endogenous GAPDH housekeeping gene. Please refer to Supplemental Table S6 for primer sequences.
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