Identification of suitable reference genes in blood samples of carcinoma lung patients using quantitative real-time polymerase chain reaction.

Abstract

Introduction: Lung cancer (LC), among all other cancers, is the leading cause of death worldwide, while the third most common cancer-causing mortality in India. Several techniques of the assay for early detection of cancer that improve survival rates have been employed in tissues and cell lines. Reverse transcriptase quantitative polymerase chain reaction (RTqPCR) is one of the most common techniques employed for gene expression studies for the normalization of a target gene using a reference gene (RG). The present study used the three most common RGs: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-Actin, and 18s ribosomal ribonucleic acid (18s rRNA), which were assessed by qPCR to validate, as of which is a more effective RG in blood samples of LC patients.

Materials and methods: A total of thirty participants with LC of non-small cell and small cell type were included along with twenty healthy controls. Ribonucleic acid (RNA) isolated from peripheral blood mononuclear cells was quantified, prepared for complementary deoxyribose nucleic acid synthesis, and analyzed for expression of three RG on RTqPCR.

Results: Expression levels as Ct values of studied RG were reported as mean ± standard deviation for GAPDH (26.97 ± 5.107), β-actin (20.5 ± 2.3), and 18s rRNA (25.10 ± 4.075). GAPDH showed the lowest expression, whereas β-actin showed the highest expression among the studied RG in subjects of LC. The expression of GAPDH and 18s rRNA were statistically significantly lower than β-actin (p < 0.0001), whereas expression levels of GAPDH and 18s rRNA were comparable. However, the expression level of only β-actin in LC patients was comparable with healthy controls with P < 0.1611 at 95% confidence interval.

Conclusion: It is concluded that β -actin may be considered the most suitable RG isolated and studied from peripheral blood mononuclear cells using RT qPCR in LC.