Quantitative PCR Analysis of Ca2+ Signaling Genes

qPCR was performed on a Roche LightCycler 480 System (Roche) using the 5′ nuclease assay (63 (link)). Total RNA was isolated using TRIzol (Life Technologies Hungary Ltd), DNase treatment was performed according to the manufacturer's protocol, and then 1 μg of total RNA was reverse-transcribed into complementary DNA using High-Capacity cDNA Kit from Life Technologies Hungary Ltd. PCR amplification was performed using the TaqMan Gene Expression Assays (assay IDs: Mm01175211_m1 for RYR1, Mm00465877_m1 for RYR2, Mm01328421_m1 for RYR3, Mm00439907_m1 for inositol 1,4,5-trisphosphate receptor (ITPR) type 1, Mm00444937_m1 for ITPR type 2, Mm01306070_m1 for ITPR type 3, Mm00613173_m1 for TRPM4, Mm01129032_m1 for TRPM5, and Mm00444690_m1 for TRPC3) and the TaqMan universal PCR master mix protocol (Applied Biosystems). As internal control, transcripts of the housekeeping gene (GAPDH; assay ID: Mm99999915_g1) were determined. The amount of the transcripts was normalized to the housekeeping gene using the ΔCT method.

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Diszházi G., Magyar Z.É., Lisztes E., Tóth-Molnár E., Nánási P.P., Vennekens R., Tóth B.I, & Almássy J. (2021). TRPM4 links calcium signaling to membrane potential in pancreatic acinar cells. The Journal of Biological Chemistry, 297(3), 101015.

Publication 2021

LightCycler 480 System TRIzol High-Capacity cDNA Kit TaqMan Gene Expression Assays TaqMan universal PCR master mix protocol

Assay Cdna Dnase Gapdh Gene expression Housekeeping gene Inositol Ryr1 Ryr2 Ryr3 Trizol Trpm5

Corresponding Organization : University of Debrecen

Other organizations : University of Szeged, VIB-KU Leuven Center for Brain & Disease Research

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Variable analysis

independent variables

RYR1 expression
RYR2 expression
RYR3 expression
ITPR type 1 expression
ITPR type 2 expression
ITPR type 3 expression
TRPM4 expression
TRPM5 expression
TRPC3 expression

dependent variables

Transcript levels of RYR1, RYR2, RYR3, ITPR type 1, ITPR type 2, ITPR type 3, TRPM4, TRPM5, and TRPC3

control variables

Housekeeping gene GAPDH expression

positive controls

Not specified

negative controls

Not specified

Annotations

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