Quantifying Gene Expression in Yeast

RNA was extracted from cells grown in YPD using a RiboPure RNA Purification Kit for Yeast (Ambion, Invitrogen). cDNA was synthesized from DNase I-treated RNA using Superscript III Reverse Transcriptase (Invitrogen) and qPCR was performed using KAPA SYBR Fast Universal qPCR Kit (KAPA Biosystems) with primers listed Table 3. Quantification was performed using the 2^–ΔΔCT method and expression was normalized to TAF10 (Livak and Schmittgen, 2001 (link); Teste et al., 2009 (link)).

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Masser A.E., Kang W., Roy J., Mohanakrishnan Kaimal J., Quintana-Cordero J., Friedländer M.R, & Andréasson C. (2019). Cytoplasmic protein misfolding titrates Hsp70 to activate nuclear Hsf1. eLife, 8, e47791.

Publication 2019

RiboPure RNA Purification Kit for Yeast Superscript III Reverse Transcriptase KAPA SYBR Fast Universal qPCR Kit

Cdna Dnase I rna Primers Reverse transcriptase Rna i Yeast

Corresponding Organization :

Other organizations : Stockholm University, Science for Life Laboratory

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Protocol cited in 2 other protocols

Variable analysis

independent variables

None explicitly mentioned

dependent variables

Gene expression

control variables

YPD growth medium
RiboPure RNA Purification Kit for Yeast (Ambion, Invitrogen)
DNase I treatment
Superscript III Reverse Transcriptase (Invitrogen)
KAPA SYBR Fast Universal qPCR Kit (KAPA Biosystems)
TAF10 as reference gene for normalization

controls

Positive control: None mentioned
Negative control: None mentioned

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