Construction of a porcine skeletal muscle cDNA microarray and its application on the identification of differentially expressed genes in different biological contexts

Gene expression profiling using DNA microarrays has the potential to illuminate the molecular processes that govern the phenotypic characteristics of porcine skeletal muscles, such as hypertrophy or atrophy, and the expression of specific fibre types. This information is not only important for understanding basic muscle biology but also provides underpinning knowledge for enhancing the efficiency of livestock production. This thesis describes the de novo development of a composite skeletal muscle cDNA microarray, comprising 5,500 clones from two developmentally distinct cDNA libraries (longissimus dorsi of a 50-day porcine foetus and the gastrocnemius of a 3-day-old pig). The cDNA clones selected for the microarray assembly were of low to moderate abundance, as indicated by colony blot hybridisation. Once constructed, the porcine cDNA microarray was used to profile the differential expression of genes between the psoas (red muscle) and the longissimus dorsi (white muscle) of a pig, by co-hybridisation of Cy3 and Cy5 labelled cDNA derived from these two muscles. Clones that were preferentially more highly expressed in one muscle type were chosen for identification by sequencing. A number of novel candidate regulatory genes and candidate genes that could be involved in muscle phenotype determination were identified. Gene expression results from seven microarray slides (replicates) correctly identified genes (e.g., genes of mitochondrial origin, genes for myosin heavy chain fast isoforms) that were expected to be differentially expressed, as well as a number of candidate regulatory genes (e.g., genes for bin 1, heat shock cognate protein, casein kinase 2 al subunit). A novel gene kc2725 was also identified as being differentially expressed. These candidate genes could be involved in muscle phenotype determination, and include several members of the casein kinase 2 signalling pathways (e.g., casein kinase 2 alphal subunit, small muscle protein, tyrosine kinase 9-like A6-related protein). Quantitative real-time RT-PCR performed on the selected genes (e.g., bin 1, novel gene kc2725, myosin heavy chain lib) was used to confirm the results from the microarray. The red-white muscle microarray expression analysis demonstrated the effectiveness of the porcine cDNA microarray for high throughput differential gene expression. Differential gene expression using the porcine skeletal muscle cDNA microarray was also studied in the ionomycin-treated and the control vehicle-treated porcine skeletal muscle myocytes by co-hybridisation of Cy3 and Cy5 labelled cDNA targets derived from both cell groups. Results from eight replicated microarray hybridisations profiled high throughput gene expression and identified a number of ionomycin-regulated genes (e.g., genes for protein tyrosine phosphatase non-receptor type 13, dystrophia myotonica-protein kinase) in porcine skeletal muscle. This study further confirmed effectiveness of the porcine cDNA microarray for profiling differential gene expression

Development of a porcine skeletal muscle cDNA microarray: analysis of differential transcript expression in phenotypically distinct muscles

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background Microarray profiling has the potential to illuminate the molecular processes that govern the phenotypic characteristics of porcine skeletal muscles, such as hypertrophy or atrophy, and the expression of specific fibre types. This information is not only important for understanding basic muscle biology but also provides underpinning knowledge for enhancing the efficiency of livestock production. Results We report on the de novo development of a composite skeletal muscle cDNA microarray, comprising 5500 clones from two developmentally distinct cDNA libraries (longissimus dorsi of a 50-day porcine foetus and the gastrocnemius of a 3-day-old pig). Clones selected for the microarray assembly were of low to moderate abundance, as indicated by colony hybridisation. We profiled the differential expression of genes between the psoas (red muscle) and the longissimus dorsi (white muscle), by co-hybridisation of Cy3 and Cy5 labelled cDNA derived from these two muscles. Results from seven microarray slides (replicates) correctly identified genes that were expected to be differentially expressed, as well as a number of novel candidate regulatory genes. Quantitative real-time RT-PCR on selected genes was used to confirm the results from the microarray. Conclusion We have developed a porcine skeletal muscle cDNA microarray and have identified a number of candidate genes that could be involved in muscle phenotype determination, including several members of the casein kinase 2 signalling pathway.Peer Reviewe

Detection of RNA-binding Proteins by RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs' as well as long non-coding RNAs' (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor's (AR) 3' untranslated region (3'UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.NMRC (Natl Medical Research Council, S’pore)Published versio