Muscle deterioration as a physiological response to the energetic demands of fish spawning represents a unique model for studying the mechanisms of muscle degradation in non-mammalian animals. A salmonid microarray, containing 16,006 cDNAs, was used to study the transcriptome response to atrophying fast-switch muscles from gravid rainbow trout in comparison to sterile fish. Total RNA was isolated using the Trizol reagent (Invitrogen) according to manufacturer’s instruction. Concentrations of isolated RNA were determined by measuring absorbance at 260 nm. The integrity of RNA was determined by agarose gel electrophoresis. Poly(A)+ RNA was purified using Oligotex mRNA Mini Kit (Qiagen) according to manufacturer’s instruction. A salmonid microarray containing cDNAs representing 16,006 genes selected from atlantic salmon and rainbow trout expressed sequence tag databases was used to profile changes of gene expression in spawning-induced atrophying muscles. mRNA (0.8 μg) from trout muscles were used as templates in reverse transcription reactions incorporating amino-allyl dUTP into the cDNA using oligo-d(T) primer and Superscript II reverse transcriptase (Invitrogen). The synthesized cDNAs from atrophying (fertile fish) and non-atrophying muscles (sterile fish) were differentially labeled using N-hydroxysuccinate-derived Cy3 and Cy5 dyes, respectively (GE Health). Labeled cDNAs were purified using a PCR purification kit (Qiagen) to remove unincorporated dyes. The Cy3 and Cy5 labeled cDNAs were then combined and concentrated down to 20 μl using a Microcon-30 concentrator (Millipore) followed by addition of 130 µl of Slidehyb 3 of solution (Ambion, Inc. Austin, TX). Microarray hybridizations were performed on a Tecan HS400 automated microarray hybridization station (Tecan US, Durham, NC). The slides were placed on the machine at 60°C for 2 minutes followed by a pre-hybridization at 55°C for 30 minutes with pre-hybridization solution (5XSSC, 1% SDS, 1% BSA) under medium agitation. After brief washing at 60°C for 1 minute, differentially labeled cDNAs in hybridization buffer (~145 µl) were injected into the hybridization chamber. The hybridizations were carried out for 3 hours at 60°C followed by another 13 hours at 55°C. Arrays were washed 2x in 2XSSC, 01% SDS, followed by 2x in 0.1XSSC, 0.1% SDS at room temperature. Following 2 more washes in 0.1XSSC, the slides were rinsed in water and dried by centrifugation.
Muscle deterioration as a physiological response to the energetic demands of fish spawning represents a unique model for studying the mechanisms of muscle degradation in non-mammalian animals. A salmonid microarray, containing 16,006 cDNAs, was used to study the transcriptome response to atrophying fast-switch muscles from gravid rainbow trout in comparison to sterile fish. Total RNA was isolated using the Trizol reagent (Invitrogen) according to manufacturer’s instruction. Concentrations of isolated RNA were determined by measuring absorbance at 260 nm. The integrity of RNA was determined by agarose gel electrophoresis. Poly(A)+ RNA was purified using Oligotex mRNA Mini Kit (Qiagen) according to manufacturer’s instruction. A salmonid microarray containing cDNAs representing 16,006 genes selected from atlantic salmon and rainbow trout expressed sequence tag databases was used to profile changes of gene expression in spawning-induced atrophying muscles. mRNA (0.8 μg) from trout muscles were used as templates in reverse transcription reactions incorporating amino-allyl dUTP into the cDNA using oligo-d(T) primer and Superscript II reverse transcriptase (Invitrogen). The synthesized cDNAs from atrophying (fertile fish) and non-atrophying muscles (sterile fish) were differentially labeled using N-hydroxysuccinate-derived Cy3 and Cy5 dyes, respectively (GE Health). Labeled cDNAs were purified using a PCR purification kit (Qiagen) to remove unincorporated dyes. The Cy3 and Cy5 labeled cDNAs were then combined and concentrated down to 20 μl using a Microcon-30 concentrator (Millipore) followed by addition of 130 µl of Slidehyb 3 of solution (Ambion, Inc. Austin, TX). Microarray hybridizations were performed on a Tecan HS400 automated microarray hybridization station (Tecan US, Durham, NC). The slides were placed on the machine at 60°C for 2 minutes followed by a pre-hybridization at 55°C for 30 minutes with pre-hybridization solution (5XSSC, 1% SDS, 1% BSA) under medium agitation. After brief washing at 60°C for 1 minute, differentially labeled cDNAs in hybridization buffer (~145 µl) were injected into the hybridization chamber. The hybridizations were carried out for 3 hours at 60°C followed by another 13 hours at 55°C. Arrays were washed 2x in 2XSSC, 01% SDS, followed by 2x in 0.1XSSC, 0.1% SDS at room temperature. Following 2 more washes in 0.1XSSC, the slides were rinsed in water and dried by centrifugation.
Label
cy5
Scan protocol
ScanArray Lite microarray scanner was used to scan arrays and ScanArray Express software (PerkinElmer, Wellesley, MA) was used to process array images, align spots, integrate robot-spotting files with the microarray images and quantify spots.
Description
A salmonid microarray, containing 16,006 cDNAs, was used to study the transcriptome response to atrophying fast-switch muscles from gravid rainbow trout in comparison to sterile fish.
Data processing
Pre-processing and normalization of data was accomplished using R-project statistical environment and Bioconductor through the GenePix AutoProcessor (GPAP) website.
