Evaluation of Protein Expression in Housekeeping Genes across Multiple Tissues in Rats

Abstract

BackgroundHousekeeping genes, which show constant protein expression patterns between different tissue types, are very important in molecular biological studies as an internal control for protein research.MethodsThe protein expression profiles of seven housekeeping genes (HPRT1, PPIA, GYS1, TBP, YWHAZ, GAPDH and ACTB) in various rat tissues (cerebrum, cerebellum, cardiac ventricle and atrium, psoas muscle, femoral muscle, liver, spleen, kidney, and aorta) were analyzed by Western blot and compared by coefficient of variation (CV).ResultsHPRT1 was stably expressed (CV≤10%) in six tissues (cerebrum, cerebellum, ventricle, femoral muscle, spleen, and kidney), PPIA was stably expressed in five tissues (cerebrum, cerebellum, ventricle, spleen and kidney), YWHAZ was stably expressed in three tissues (cerebrum, cerebellum, and kidney), and GAPDH was stably expressed in four tissues (cerebrum, ventricle, psoas muscle, and kidney). In comparison, GYS1, TBP, and ACTB were found to have CV values over 10% in all tissues. Of the seven genes examined, four (HPRT1, PPIA, YWHAZ, and GAPDH) were found to be stably expressed across multiple organs, with low CV values (≤10%).ConclusionsThese results will provide fundamental information regarding internal controls for protein expression studies and can be used for analysis of postmortem protein degradation patterns in forensic medicine.

Aging‐associated changes in hippocampal glycogen metabolism in mice. Evidence for and against astrocyte‐to‐neuron lactate shuttle

“…For detection of mRNA for metabolic genes commercially available TaqMan probes (Applied Biosystems, Waltham, Massachusetts, USA) dedicated to mouse tissue were used (see Supporting Information, Table S2). The level of mRNA for glycogen synthase ( Gys1 ) was used as reference (Kim et al, ). In contrast to Gys1 , our proteomic data revealed that the expression of all commonly used in qRT‐PCR referential genes (Boda, Pini, Hoxha, Parolisi, & Tempia, ) changed during aging or they were expressed at very low levels (Supporting Information, Table S3).…”

The role of extracellular matrix stiffness in regulating cytoskeletal remodeling via vinculin in synthetic smooth muscle cells

“…GAPDH was chosen to normalize vinculin quantity in the supernatant fraction (cytosol and membrane) across different samples because of its relatively high abundance and stable protein expression levels in tissues with different stiffness levels. [36] α-actin wasn’t used because of possible large variations in its protein expression levels over different stiffness. [36] Despite high Vcl levels at both 2 kPa and >GPa (polystyrene), vinculin protein expression level at 2 kPa in cytosol and membrane bound is significantly less than that from polystyrene (Figure 2).…”

“…[36] α-actin wasn’t used because of possible large variations in its protein expression levels over different stiffness. [36] Despite high Vcl levels at both 2 kPa and >GPa (polystyrene), vinculin protein expression level at 2 kPa in cytosol and membrane bound is significantly less than that from polystyrene (Figure 2). Another interesting observation is that as stiffness increased up to 50 kPa, Vcl level decreased but vinculin protein expression increased.…”

Identification of valid endogenous control genes for determining gene expression in C6 glioma cell line treated with conditioned medium from adipose-derived stem cell

“…However, the PCR efficiency can vary over time and across samples [49]. Therefore, the choice of an appropriate reference gene is fundamental to avoid an erroneous interpretation of the results.…”