RNA isolation and real time PCR

VP Valentina Pastori
GZ Gianluca Zambanini
EC Elisabetta Citterio
TW Tamina Weiss
YN Yukio Nakamura
CC Claudio Cantù
AR Antonella Ellena Ronchi
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Total RNA from ≥ 105 cells were purified with TRIzol Reagent (Euroclone) and retrotranscribed (High Capacity cDNA Reverse Transcription Kit, Applied Biosystem). RT-PCR analysis was performed using StepOne (Thermofisher). Specific PCR product accumulation was monitored by using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad) fuorescent dye in 12 μl reaction volume. Dissociation curves confirmed the homogeneity of PCR products. Primers, designed to amplify 150 to 300bp amplicons, are listed in the Supplementary table 3. Each experiment was done in three biological replicates and cDNAs from each replicate were analysed in technical triplicates.

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