Analysis of commonly used reference genes in the frontopolar cortex in schizophrenia and bipolar disorder
Medina AM, Hughes E, Bunney WE, Myers RM, Schatzberg AF, Barchas JD, Akil H, Watson SJ
Society for Neuroscience. 2019.
Quantitative reverse transcription PCR (qRT-PCR) is frequently used to demonstrate changes in gene expression levels in a wide range of conditions. A normalization step is routinely included, preferably by using multiple stably expressed reference genes, also known as housekeeping genes. Some of those genes, such as beta-actin (ACTB) or ubiquitin (UBC) are universally considered as unvarying and used for the normalization step. However, it is important to acknowledge the inherent biological variability of each set of samples and the possible effect of disease on the genes selected. In this study, we aim to determine what the best set of reference genes would be for the analysis of gene expression in the human Frontopolar cortex in healthy control subjects compared to bipolar disorder (BPD) and schizophrenic subjects (SZ).
Methods: Human brain blocks from the Frontopolar cortex of 23 controls, 24 schizophrenics, and 21 bipolar disorder subjects were obtained from the Brain Donor Program at the University of California, Irvine by agreement with the Pritzker Neuropsychiatric Consortium. The subjects included in the schizophrenic and bipolar disorder groups met diagnostic criteria from the Diagnostics and Statistical Manual of Mental Disorders (DSM-V). In the control group, there was no evidence of psychiatric or neurological disorders. Variables accounted for include gender, age and postmortem interval. All subjects used in the study had tissue pH above 6.5 and agonal factor scores (AFS) of zero. Fresh frozen blocks of tissue averaging 500 mg were dissected from the frontopolar region of each subject and processed for RNA extraction to be used for gene expression studies. We used TaqMan® microfluidic cards to perform qPCR in a Viia™7 machine for evaluation of the gene expression profiles of a set of 12 commonly used reference genes: 18s, ACBT, B2M, HPRT1, GUSB, HMBS, IPO8, PGK1, RPLP0, TBP, TFRC, UBC. ExpressionSuite® software version 1.1 was used to analyze the results.
Results: ExpressionSuite® software allows the evaluation of different quality control measurements for the endogenous controls and targets run in a microfluidic card. After looking at parameters such as standard deviation, Ct max, and Cq confidence values, our results show that several commonly used reference genes are not ideal for normalization of gene expression in our samples. Of twelve genes tested, only five were a good fit for our study, as the other seven showed high variability, especially in the control samples.
We suggest that a careful selection of housekeeping genes appropriate to the sample being used is a fundamental first step before proceeding with gene expression studies using qPCR.