Seleção de genes HOUSEKEEPING para análise de PCR quantitativo do gene GSTM1
Resumo
Quantitative real-time PCR (qPCR) is a technique that combines the exponential
amplification system of a DNA fragment with the capture of data at the moment the
amplification reaction occurs by means of fluorescence measurements. The qPCR became one
of the most widely used techniques when it comes to gene expression analysis because of its
precision, wide dynamic range, sensitivity, reproducibility. When it’s compared the gene
expression between different samples and / or tissues, it is paramount to consider the aspects
of the experimental variation resulting from the amount of starting material, RNA extraction
and reverse transcription efficiencies. In order to eliminate such variations it’s necessary to
use a normalizing gene to ensure the accuracy of the levels of expression of the assay qPCR.
Therefore, this study aims to select reference genes for gene expression analysis assays of
the GSTM1 family gene. It was collected peripheral blood samples (5 mL) from exposed
individuals and not exposed ones to Mercury (Hg). The RNA extraction was performed,
then cDNA synthesis and qPCR for seven normalizing genes
(ACTB, GAPDH, 18S, HPRT1, B2M, GUSB and TBP). For the analysis of normalization of
the reference genes, It was applied the technique developed by Silver et al. (2016) called
"gene pairs". The results of the present work demonstrate that
the ACTB, GAPDH and B2Mgenes presented better stability to be used as reference genes
for GSTM1 gene studies.