Yasmin Hurd | PubMed Health

PubMed2006

Validation of endogenous controls for quantitative gene expression analysis: application on brain cortices of human chronic alcoholics.

Real-time PCR is frequently used for gene expression quantification due to its methodological sensitivity and reproducibility. The gene expression is quantified by normalization to one or more reference genes, usually beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPD) or to ribosomal RNA (18S). However, different environmental or pathological conditions might also influence the expression of normalizing genes, which could severely skew the interpretation of quantitative results. This study evaluates whether 16 genes frequently used as endogenous controls in expression studies, can serve as such for comparison of human brain tissues of chronic alcoholics and control subjects. The prefrontal and motor cortices that are affected differently by chronic alcohol consumption were analyzed. The reference genes that have no or small differences in expression in alcoholics and control subjects, were found to be specific for each region: beta-actin (ACTB) and ribosomal large P0 (RPLP0) for the prefrontal cortex while importin 8 (IPO8) and RNA polymerase II (POLR2A) for the motor cortex. Four out of sixteen analyzed genes demonstrated significant differences in expression between alcoholics and controls: phosphoglycerate kinase (PGK1), hypoxanthine phosphoribosyl transferase (HPRT1) and peptidylprolyl isomerase A (PPIA) in the motor cortex and beta-2-microglobulin (B2M) in the prefrontal cortex. Our study demonstrates the importance of validation of endogenous control genes prior to real-time PCR analysis of human brain tissues. Prescribed and non-prescribed drugs, pathological or environmental conditions along with alcohol abuse may differentially influence expression of reference genes.

Adult↗

PubMed2004

Cloning and expression of the human NMDA receptor subunit NR3B in the adult human hippocampus.

The mammalian genome encodes seven different NMDA receptor subunits. All of these subunits have been cloned in the human except for NR3B. Here, we have successfully obtained two full-length clones of human NR3B using a PCR-based cloning approach. The open reading frame of the consensus sequence contains 3129 nucleotides translating into 1043 amino acids. The overall polypeptide sequence identity with mouse NR3B is 74.9%, which is lower than for the other six NMDA receptor subunits. In particular, the translated part of exon 9 is only 37.8% identical between human and mouse. The GRIN3B gene, which encodes human NR3B, maps to chromosome 19p13.3, between WDR18 and C19orf6 (membralin). Human NR3B is encoded by nine exons, as in mouse NR3B, and exon-intron boundaries are conserved between the species. However, exon 9 is substantially longer in the human. In situ hybridization data shows that NR3B mRNA is expressed in the human hippocampal formation (CA1-CA4 and dentate gyrus) and adjacent neocortex. The expression of NR3A mRNA was restricted to the dentate gyrus and layers IV and V of the neocortex. Our results may have implications for the understanding of the role of NMDA receptors for physiological and pathological processes in these forebrain regions.

Aged↗