Quantitative Gene Expression Analysis via qPCR

Gene expression was evaluated via quantitative real-time polymerase chain reaction (qPCR) according to standard protocols previously reported by our group (Pacheco et al., 2014 (link); Morais et al., 2016 (link)). Briefly, after cell treatment, the total RNA was extracted using the RNeasy kit (Qiagen, Netherlands) followed by DNase treatment, Then, cDNA strands were constructed using a SuperScript^® III First-Strand Synthesis kit (InvitrogenTM, Life Technologies Inc., United States), according to manufacturer’s instructions. Finally, the samples were applied in a SYBR^® green (Applied Biosystems, United States)-based reaction with specific and validated primers to evaluate mRNA levels of genes coding for dynein chains. RNA levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and quantified using the Livak 2^−ΔΔCT method (Livak and Schmittgen, 2001 (link)). The experiments were conducted by Step One Plus^® PCR real-time system (Applied Biosystems, United States).

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Morais K.L., Ciccone L., Stura E., Alvarez-Flores M.P., Mourier G., Driessche M.V., Sciani J.M., Iqbal A., Kalil S.P., Pereira G.J., Marques-Porto R., Cunegundes P., Juliano L., Servent D, & Chudzinski-Tavassi A.M. (2023). Structural and functional properties of the Kunitz-type and C-terminal domains of Amblyomin-X supporting its antitumor activity. Frontiers in Molecular Biosciences, 10, 1072751.

Publication 2023

RNeasy kit SuperScript® III First-Strand Synthesis kit SYBR® green Step One Plus® PCR real-time system

Cdna Cell Dnase Dynein Gene expression Genes Glyceraldehyde 3 phosphate dehydrogenase Mrna Primers Quantitative real time polymerase chain reaction Step one Sybr green Synthesis

Corresponding Organization : Technologies pour la Santé

Other organizations : Universidade Federal de São Paulo, University of Pisa

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Variable analysis

independent variables

Cell treatment

dependent variables

MRNA levels of genes coding for dynein chains

control variables

GAPDH mRNA levels for normalization

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