ABSTRACT
BACKGROUND: Quantitative real time reverse transcription PCR (qRT-PCR) is one of the most important techniques for gene-expression analysis in molecular based studies. Selecting a proper internal control gene for normalizing data is a crucial step in gene expression analysis via this method. The expression levels of reference genes should be remained constant among cells in different tissues. However, it seems that the location of cells in different tissues might influence their expression. The purpose of this study was to determine whether the source of mesenchymal stem cells (MSCs) has any effect on expression level of three common reference genes (GAPDH, ß-actin and ß2-microglobulin) in equine marrow- and adipose- derived undifferentiated MSCs and consequently their reliability for comparative qRT-PCR. MATERIALS AND METHODS: Adipose tissue (AT) and bone marrow (BM) samples were harvested from 3 mares. MSCs were isolated and cultured until passage 3 (P3). Total RNA of P3 cells was extracted for cDNA synthesis. The generated cDNAs were analyzed by quantitative real-time PCR. The PCR reactions were ended with a melting curve analysis to verify the specificity of amplicon. RESULTS: The expression levels of GAPDH were significantly different between AT- and BM- derived MSCs (p < 0.05). Differences in expression level of ß-actin (P < 0.001) and B2M (P < 0.006.) between MSCs derived from AT and BM were substantially higher than GAPDH. In addition, the fold change in expression levels of GAPDH, ß-actin and B2M in AT-derived MSCs compared to BM-derived MSCs were 2.38, 6.76 and 7.76, respectively. CONCLUSION: This study demonstrated that GAPDH and especially ß-actin and B2M express in different levels in equine AT- and BM- derived MSCs. Thus they cannot be considered as reliable reference genes for comparative quantitative gene expression analysis in MSCs derived from equine bone marrow and adipose tissue.
ABSTRACT
Flow cytometry is a widely used application for validating the accuracy of sperm sexing. However, this method is relatively expensive and requires considerable technical support. An alternative method employing simpler technology at low cost could be suitable for the evaluation of bovine semen in laboratories with low budgets. We used a SYBR Green Real-Time PCR assay to determinate sex ratio in bovine semen. The PLP and SRY genes were amplified to isolate the specific fragments of X- and Y-chromosome sequences, respectively. Two certified standard curves were obtained using two plasmids containing PLP and SRY amplicons. Our results show no significant difference in semen sex ratio in unsorted semen (54.7±0.52% X and 47.6±0.60% Y). However, significant difference was observed in X/Y-sorted semen (93.3±0.08% X and 91.4±0.06% Y-sperm), as compared to the expected ratio in unsorted semen or the post-sorting reanalysis data. The evolution of X-chromosome bearing sperm content in unsorted samples showed an average of 52.6 for ejaculates and 51.8 for the commercial semen. In order to confirm our results, the accuracy, repeatability and reproducibility of the method were tested resulting in 98.2% accuracy, repeatability of CV=5.59% and reproducibility of CV=5.40%. Thus, this method is demonstrated to be a reliable and inexpensive way to test sexual chromosome content in semen samples.
