Short Communication: Optimization of semi-quantitative RT PCR analysis for CPT I gene expression in Rainbow trout (Oncorhynchus mykiss)
A key enzyme in mitochondrial βoxidation, carnitine palmitoyltransferase (CPT) I, is transcriptionally regulated in mammals, but this enzyme also experiences allosteric modulations (Harano et al., 1985; Murthy and Pande, 1987; Bezaire et al., 2004). CPT I is located on the inner side of the outer mitochondrial membrane and catalyses the conversion of acyl-CoA to fatty acylcarnitine (Kerner and Hoppel, 2000; Price et al., 2000). Quantitative RT-PCR is a reliable technique for measuring transcripts in small amounts of tissue (Spriewald et al., 2000). With this technique, multiple mRNAs can be assayed simultaneously in a relatively short period of time. Here we describe the standard procedure, optimized in our laboratory, to assess CPT I levels with βactin as an internal control in rainbow trout, and all the necessary controls to ensure a quantitative analysis.
| Main Authors: | , , , |
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| Format: | Journal Contribution biblioteca |
| Language: | English |
| Published: |
2011
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| Subjects: | Oncorhynchus mykiss, Reverse Transcription, Primers, Carnitine Palmitoyltransferase I, Rainbow Trout, RT PCR, Gene Expression, Optimization, Gene, |
| Online Access: | http://hdl.handle.net/1834/11425 |
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| Summary: | A key enzyme in mitochondrial βoxidation, carnitine palmitoyltransferase (CPT) I, is transcriptionally regulated in mammals, but this enzyme also experiences allosteric modulations (Harano et al., 1985; Murthy and Pande, 1987; Bezaire et al., 2004). CPT I is located on the inner side of the outer mitochondrial membrane and catalyses the conversion of acyl-CoA to fatty acylcarnitine (Kerner and Hoppel, 2000; Price et al., 2000). Quantitative RT-PCR is a reliable technique for measuring transcripts in small amounts of tissue (Spriewald et al., 2000). With this technique, multiple mRNAs can be assayed simultaneously in a relatively short period of time. Here we describe the standard procedure, optimized in our laboratory, to assess CPT I levels with βactin as an internal control in rainbow trout, and all the necessary controls to ensure a quantitative analysis. |
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