Gene Expression Quantification by qRT-PCR

Measurement of gene expression by qRT-PCR was performed as previously outlined^{43 (link)}. Briefly, total RNA was isolated using Trizol or Direct-zol (Zymo Research) and used for cDNA synthesis (2 μg liver RNA or 1–2 μg cell RNA) by two-step RT-PCR with the High Capacity cDNA synthesis kit (Roche). Levels of mRNA were semi-quantified with Sensifast SYBR Green mix (Bioline) and gene-specific primers (Integrated DNA Technologies) and were normalized to the housekeeping gene Ppia (CypA). For specific primer sequences, see Supplementary Table 1.

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Taddeo E.P., Hargett S.R., Lahiri S., Nelson M.E., Liao J.A., Li C., Slack-Davis J.K., Tomsig J.L., Lynch K.R., Yan Z., Harris T.E, & Hoehn K.L. (2017). Lysophosphatidic acid counteracts glucagon-induced hepatocyte glucose production via STAT3. Scientific Reports, 7, 127.

Publication 2017

Trizol Direct-zol High Capacity cDNA synthesis kit Sensifast SYBR Green mix gene-specific primers

Cdna Cell Gene Gene expression Housekeeping gene Liver Mrna Primer Rt pcr Sybr green Synthesis Trizol

Corresponding Organization : UNSW Sydney

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Levels of mRNA

control variables

Housekeeping gene Ppia (CypA)

controls

Positive control: None mentioned
Negative control: None mentioned

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