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

Bai, Qianfan (2004) Construction of a porcine skeletal muscle cDNA microarray and its application on the identification of differentially expressed genes in different biological contexts. PhD thesis, University of Glasgow.

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Abstract

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.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Kin-Chow Chang
Keywords: Genetics, Animal sciences
Date of Award: 2004
Depositing User: Enlighten Team
Unique ID: glathesis:2004-71180
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 10 May 2019 10:49
URI: http://theses.gla.ac.uk/id/eprint/71180

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