Quantitative Analysis of Gene Expression

Total RNA was isolated from cell lysates using NucleoZol (TaKaRa Bio) according to manufacturer’s instructions, and 1 μg of total RNA was suspended in 10 μl of RNase-free water. RT-qPCR was performed with 1 μl of 1:10 dilution of the RNA using Luna Universal One-Step RT-qPCR kit (New England BioLabs), with a total reaction volume of 10 μl in a Bio-Rad CFX384 Real-Time System (Bio-Rad). Primers used for Ago2 [90 (link)], Dicer [72 (link)], GPRC5A [62 (link)], SUMO3 [91 (link)] and SUMO1, SUMO2, and β-actin [23 (link)] transcripts are as previously described. The relative mRNA expression was derived from 2^-ΔΔCT by use of the comparative threshold cycle (CT) method. The abundance of mRNA in each sample was normalized to the amount of actin mRNA.

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Campbell A.M., De La Cruz-Herrera C.F., Marcon E., Greenblatt J, & Frappier L. (2022). Epstein-Barr Virus BGLF2 commandeers RISC to interfere with cellular miRNA function. PLoS Pathogens, 18(1), e1010235.

Publication 2022

NucleoZol Luna Universal One-Step RT-qPCR kit Bio-Rad CFX384 Real-Time System

Actin Ago2 Cell Dicer Dilution Gprc5a Mrna Primers Rnase Sumo1

Corresponding Organization : University of Toronto

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Relative mRNA expression of Ago2, Dicer, GPRC5A, SUMO3, SUMO1, and SUMO2

control variables

Amount of total RNA used (1 μg)
Dilution of RNA (1:10)
Total reaction volume (10 μl)
RT-qPCR platform (Bio-Rad CFX384 Real-Time System)
Normalization to β-actin mRNA abundance

controls

No positive or negative controls were explicitly mentioned in the protocol.

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