Quantitative RT-PCR Protocol for Gene Expression

qRT-PCR was carried out as described previously (20 (link)) in a CFX98 multicolor system (Bio-Rad) using 2 μg of total RNA reverse transcribed with an RT-PCR kit (Bio-Rad). Single-product amplification was confirmed by melting-curve analysis, and primer efficiency was near 100% in all experiments. Quantifications are expressed in arbitrary units, and target mRNA abundance was normalized to the expression of GAPDH by the method of Pfaffl (52 (link)). All the qRT-PCR results are representative of at least three independent experiments (n > 3). A list of primers is provided in the supplemental material.

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A.P. S, & Laishram R.S. (2018). Nuclear Phosphatidylinositol-Phosphate Type I Kinase α-Coupled Star-PAP Polyadenylation Regulates Cell Invasion. Molecular and Cellular Biology, 38(5), e00457-17.

Publication 2017

CFX98 multicolor system RT-PCR kit

Gapdh Mrna Primers Rt pcr

Corresponding Organization : Rajiv Gandhi Centre for Biotechnology

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Target mRNA abundance

control variables

Total RNA reverse transcribed with an RT-PCR kit (Bio-Rad)
GAPDH expression (used for normalization)

controls

Positive control: Single-product amplification confirmed by melting-curve analysis
Primer efficiency: near 100% in all experiments

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