RNA was isolated using TRIzol reagent (Invitrogen) and used in a cDNA synthesis reaction with random hexamers (Promega). cDNA was used as a template in quantitative PCR reactions using a FAST SYBR mix (KAPA Biosystems) on an Eppendorf Realplex with Mbd3, Smarca4, or GAPDH specific primers (Hainer et al., 2015 (link)).
Fast sybr mix
Manufactured by Roche
FAST SYBR mix is a ready-to-use solution for real-time PCR applications. It contains the necessary components for sensitive and specific detection of DNA sequences, including a SYBR Green I-based fluorescent dye and a thermostable DNA polymerase.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using fast sybr mix
1
RNA Isolation and qPCR Analysis
2
Quantitative RT-PCR with RNA Extraction
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RNA was isolated using TRIzol reagent (Invitrogen). Total RNA was treated with DNase I (New England Biolabs) for 15 min at 37°C. One microgram of RNA was used in a cDNA synthesis reaction using either random hexamers (Promega), oligo-dT, or strand-specific primers (see Supplemental Table S1) where indicated, with reverse transcriptase or a no-RT control. cDNA was used as a template in qPCR reactions using a FAST SYBR mix (KAPA Biosystems) on an Eppendorf Realplex with specific primers (see Supplemental Table S1). GAPDH was used as a loading control for randomly primed and oligo-dT-amplified cDNA RT-qPCR reactions. For strand-specific reactions, cDNA was prepared in parallel with random hexamers, and a GAPDH loading control was performed.
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