Profiling and validation of reference genes for Real-Time PCR in the chronic conditions related to obesity and metabolic syndrome

Rohini Mehta, Aybike Biredinc, Reem Alhussain, Arian Afendy, Vikas Chandhoke, Zobair M. Younossi, Ancha Baranova

This is a collaborative project between

School of Systems Biology, College of Science,George Mason University, Fairfax, VA

Translational Reseach Institute, Inova Hospital, VA

So far, we completed validation of reference genes for human omental adipose samples and samples of human stomach.

Manuscript ”Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples” received prestigeous “HIGHLY ACCESSED” status in BMC Molecular Biology. We recommend ACTB and RPII as stable reference genes most suitable for gene expression studies of human visceral adipose tissue.

Manuscript "Profiling and Validation of Reference Genes in the Gastric Tissue of Morbidly Obese Patients" Is accepted for publication in Russ. J. of Mol. Biol (Mol Biol+). We recommend GAPDH and HPRT1 as stable reference genes most suitable for gene expression studies of human non-malignant gastric tissue.

BACKGROUND AND RATIONALE:

With the advances in technology se of the high throughput platforms became increasingly prevalent method of preclinical research. Currently, a RealTime-PCR, also called quantitative PCR (qPCR) is the gold standard for the detection and quantification of gene expression. However, the performance of this method is entirely dependent on the choice of internal control genes, known as reference genes or housekeeping genes. The use of housekeeping genes allows normalization of the variation in expression levels between test and control samples. The normalization is a prerequisite for accurate RT-PCR expression profiling. Until recently, there has been a pervasive assumption that all housekeeping genes behave similarly in all tissues. In recent years researchers have shown that this indeed is not the case.

A recent study testing the influence of obesity and type 2 diabetes (T2D) on the expression levels of ten housekeeping genes in human omental and subcutaneous adipose tissue  found that 18S rRNA showed the most stable expression levels in both types of fat regardless of obesity and diabetes status, where as GAPD was seen the have the highest expression level variation leading the scientists to preclude the use of GAPDH in the study of adipose tissue in studies relating to obesity and T2D (Catalán V et al. 2007). On the other hand, the performance of 18S gene is unsatisfactory as it is expressed at approximately 10000 higher levels than protein coding genes of interests. Because of that, its use requires a serial dilution of the mRNA samples which is extremely prone to the errors and, therefore, should be avoided at all costs.

The lack of stable reference gene expression is a pervasive problem. In a related study of human hepatocellular carcinoma samples six genes were tested in their capacity as stable house keeping genes: beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), succinate dehydrogenase complex, subunit A (SDHA) and ubiquitin C (UBC). Researchers observed that HMBS and GAPDH showed the least variation in expression between paired tumoral and adjacent non-tumoral tissues from HCC. Where as the combined use of HMBS, B2M, SDHA and GAPDH was found to be a stable reference set for the liver cancer cell lines: Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182 (Cicinnati VR et al. 2008). In conclusion, the study described above provided a reference standard for future qPCR experiments in of HCC pathogenesis.

To date, no such study was performed for non-cancerous chronic liver condition, particularly, liver fibrosis, steatosis and NASH. Study of obesity and T2D described above returned unsatisfactory results due to the flaws in its design, particularly, poorly chosen set of reference genes. Therefore it is vital to investigate sets of adipose and liver samples and to validate reference gene or set of genes that will be used in qPCR experiments relevant to T2D, obesity and non-cancerous liver conditions. This study will represent a crucial step in assessing the quality of data obtained from high throughput studies, improving the significance of the detected outcomes and, among other things, will opens up the possibility of studying the biological relevance of small expression differences in chronic liver diseases.

STUDY AIMS:

This research proposes to profile a set of 15 samples of the liver tissue previously collected from the patients with histologically confirmed steatosis, NASH, fibrosis, cirrhosis and normal biopsy and 10 samples of adipose tissue previously collected from morbidly obese and lean patients for an expression of a set of reference genes. Reference gene set includes: 18S, beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), succinate dehydrogenase complex, subunit A (SDHA), ubiquitin C (UBC), Beta actin (ACTB), Tyrosine 3-monooxygenase/tryptophan 5- monooxygenase activation protein, zeta polypeptide (YWHAZ), RNA polymerase II polypeptide (RP II) and TATA box binding protein (TBP). Each tissue subset (liver and adipose) will be used to determine a set of reference genes with the least variation between subgroups. These genes will then be cross-validated in a subset of samples and variability levels will be calculated.