The PFA-AMeX method achieves a good balance between the morphology of tissues and the quality of RNA content in DNA microarray analysis with laser-capture microdissection samples

Recently, large-scale gene expression profiling is often performed using RNA extracted from unfixed frozen or formalin-fixed paraffin embedded (FFPE) samples. However, both types of samples have drawbacks in terms of the morphological preservation and RNA quality. In the present study, we investigated 30 human prostate tissues using the PFA-AMeX method (fixation using paraformaldehyde (PFA) followed by embedding in paraffin by AMeX) with a DNA microarray combined with laser-capture microdissection. Morphologically, in contrast to the case of atypical adenomatous hyperplasia, loss of basal cells in prostate adenocarcinomas was as obvious in PFA-AMeX samples as in FFPE samples. As for quality, the loss of rRNA peaks 18S and 28S on the capillary electropherograms from both FFPE and PFA-AMeX samples showed that the RNA was degraded equally during processing. However, qRT-PCR with 3’ and 5’ primer sets designed against human beta-actin revealed that, although RNA degradation occurred in both methods, it occurred more mildly in the PFA-AMeX samples. In conclusion, the PFA-AMeX method is good with respect to morphology and RNA quality, which makes it a promising tool for DNA microarrays combined with laser-capture microdissection, and if the appropriate RNA quality criteria are used, the capture of credible GeneChip data is well over 80% efficient, at least in human prostate specimens.     

Quantitative assessment of muscle mass and gene expression analysis in dogs with glucocorticoid-induced muscle atrophy

The present study aimed to quantitatively evaluate muscle mass and gene expression in dogs with glucocorticoid-induced muscle atrophy. Five healthy beagles received oral prednisolone for 4 weeks (1 mg/kg/day), and muscle mass was then evaluated via computed tomography. Histological and gene expression analyses were performed using biopsy samples from the biceps femoris before and after prednisolone administration. The cross-sectional area of the third lumbar paraspinal and mid-femoral muscles significantly decreased after glucocorticoid administration (from 27.5 ± 1.9 to 22.6 ± 2.0 cm² and from 55.1 ± 4.7 to 50.7 ± 4.1 cm², respectively; P<0.01). The fast- and slow-twitch muscle fibers were both atrophied (from 2,779 ± 369 to 1,581 ± 207 μm² and from 2,871 ± 211 to 1,971 ± 169 μm², respectively; P<0.05). The expression of the growth factor receptor-bound protein 10 (GRB10) significantly increased after prednisolone administration (P<0.05). Because GRB10 suppresses insulin signaling and the subsequent mammalian target of rapamycin complex 1 activity, increased expression of GRB10 may have resulted in a decrease in protein anabolism. Taken together, 1 mg/kg/day oral prednisolone for 4 weeks induced significant muscle atrophy in dogs, and GRB10 might participate in the pathology of glucocorticoid-induced muscle atrophy in canines.     

Methyl donors dietary supplementation to gestating sows diet improves the growth rate of offspring and is associating with changes in expression and DNA methylation of insulin‐like growth factor‐1 gene

The study aimed to investigate the effects of maternal dietary methyl donors on the performance of sows and their offspring, and the associated hepatic insulin‐like growth factor‐1 (IGF‐1) expression of the offspring. A total of 24 multiparous sows were randomly fed the control (CON) or the CON diet supplemented with methyl donors (MD) at 3 g/kg betaine, 15 mg/kg folic acid, 400 mg/kg choline and 150 μg/kg VB₁₂, from mating until delivery. After farrowing, sows were fed a common lactation diet through a 28‐days lactation period and six litters per treatment were selected to be fed until at approximately 110 kg BW. Maternal MD supplementation resulted in greater birthweight (p < 0.05) and increased the piglet weights (p < 0.01) and litter weights (p < 0.05) at the age of day 28, compared with that in CON group. The offspring pigs in the MD group had greater ADG (p < 0.05) and tended to lower F:G ratio (p = 0.07) compared with that of CON group from day 28 to 180 of age. The offspring pigs from MD group had greater serum IGF‐1 concentrations and expressions of hepatic IGF‐1 gene and muscular IGF‐1 receptor (IGF‐1r) protein at birth (p < 0.05), and greater hepatic IGF‐1 protein (p = 0.03) and muscular IGF‐1r gene expressions (p < 0.05) at slaughter, than that from the CON group. Moreover, the methylation at the promoter of IGF‐1 gene in the liver of newborn piglets and finishing pigs was greater in the MD group than that of the CON group (p < 0.05). In conclusion, maternal MD supplementation throughout gestation could enhance the birthweight and postnatal growth rate of offspring, associated with an increased expression of the IGF‐1 gene and IGF‐1r, as well as the altered DNA methylation of IGF‐1 gene promotor.