Methods
Mice: Four 60 day old male mice were used for the study. The mice were obtained from Jackson labs, C57bl6/6J.
Denervation: The mice were anesthetized with Metafane, and the fur over the right thigh was shaved. An incision was made at mid thigh to expose the sciatic nerve. The right sciatic nerve was sectioned using fine scissors. The incision was closed with tissue glue, and the mice allowed to recover.
Tissue harvesting: 4 days after denervation the mice were euthanized via cervical dislocation. The right medial gastrocnemius muscle was dissected and labeled as ‘Denervted’. The contralateral left medial gastrocnemius muscle was dissected and served as the control. The muscles were frozen in liquid nitrogen. This animal procedure has been approved by KUMC IACUC.
RNA isolation: Total RNA was isolated by Trizol reagent, followed by purification with Qiagen RNA isolation Kit. RNA analysis: RNA quality was assessed on denaturing agarose gels containing formaldehyde. The three mice with the highest quality RNA in both denervated and control muscles were then subjected to microarray analysis.
Probe production: Total RNA is reversed transcribed using an oligo dT primer coupled with a T7 promoter and the Superscript Choice System (Invitrogen Live Technologies). Invitro-transcription and biotin labeling of the cRNA target is conducted using the Enzo RNA Transcript Labeling Kit (Enzo Diagnostics). The biotin labeled cRNA is fragmented prior to hybridization. The Affymetrix genechips were analyzed using the Agilent 2100 Bioanalyzer.
Statistical Analysis of Change in Gene Expression in Denervated Muscle > >