مقالات پذیرفته شده در سومین کنگره بین المللی زیست پزشکی
Validation of Suitable Housekeeping Genes for Real‐Time PCR Normalization in Human Adipose Tissue
Validation of Suitable Housekeeping Genes for Real‐Time PCR Normalization in Human Adipose Tissue
Alireza Bahiraee,1,*Solaleh Emamgholipour,2Reyhane Ebrahimi,3
1. Department of Medical Genetics, Faculty of Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran. 2. Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. 3. Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Introduction: Several studies suggested that beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S rRNA are expressed constitutively and contribute to the fundamental reference actions essential for cell viability and maintenance. However, there are inconsistency in this regard. Hence, we aimed to evaluate the accuracy of these three potential reference genes for Real‐Time quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR) application for normalization in two types of human adipose tissues.
Methods: The study was conducted on 39 women from who referred to Erfan, Loqman Hakim, and Sina hospitals, Tehran, Iran for bariatric surgery (Roux-en-Y gastric bypass and vertical sleeve gastrectomy) or elective cholecystectomy and inguinal hernia, all women aged between 20 to 53 years old including 20 obese subjects (BMI ≥35 kg/m2), and 19 normal-weight subjects (BMI ≤ 25 Kg/m2). Subcutaneous and visceral adipose tissue were collected during the specific surgeries from the participants. After homogenization of subcutaneous and visceral adipose tissue samples separately, the total RNA was extracted by RNeasy Lipid Tissue Mini Kit (Qiagen GmbH, Germany), based on the manufacturer’s protocol. Next, 1µg of total RNA was aplied as a template for first-strand complementary DNA (cDNA) synthesis by PrimeScript 1st Strand cDNA Synthesis kit (Takara, Japan) according to the manufacturer’s protocol. Finally, RT-qPCR was applied to determine the expression levels of beta-actin, GAPDH, and 18S rRNA.
Results: The gene expression level of beta-actin, GAPDH, and 18S rRNA was essentially the same in the subcutaneous and visceral fat tissues of all participants (P>0.05). Hence, all considered housekeeping genes displayed high expression stability and the analysis revealed that normalization to all of these three housekeeping genes gave a result that satisfactorily reflected the acceptable mRNA expression levels in adipose tissues.
Conclusion: Altogether, our study indicates that normalization to all beta-actin, GAPDH, and 18S rRNA housekeeping genes gave reliable results in both subcutaneous and visceral adipose tissue when comparing normal-weight and obese subject. Then, these housekeeping genes should be the preferred reference genes for normalization when studying gene expression in human adipose tissue.