RNA extraction: From cryosections from the belly portion of the muscle wit RNeasy mini kit (Qiagen AG, SWITZERLAND).
Label
[alpha-32P]dATP
Label protocol
Target labeling: reverse transcription of 4 microg total RNA with gene specific primers @50°C using Superscript RT (Life Technologies AG, Basel, SWITZERLAND), [alpha-32P]dATP, and S-modified dCTP (Ambion (Europe) Ltd, Cambridgeshire, UK).
Hybridization protocol
Annealing: 16h @ 68°C in ExpressHyb (Clontech Laboratories, Inc., Ozyme, FRANCE). Washing: 4x in (2xSSC, 1% SDS) @68°C for 1h. 1x in (0.1xSSC, 0.5% SDS) @68°C for ½ h, 1x in (2x SSC) @room temperature for 5min.
Scan protocol
Exposure: Phosphoimager screen, 4-10 daysHard & Software: Phosphorimager #425E and ImageQuant v. 3.3 (Molecular Dynamics, Sunnyvale, California, USA). Image analysis: For each spot, the pixel sum was determined with AIDA software (Raytest Schweiz AG, Urdorf, Switzerland). Background was determined from the mean of empty areas on predefined areas of the filter
Description
see other fields
Data processing
Signals were background corrected and normalized to the signal of all mRNAs on the filter (all signals except 18S rRNA and 28S rRNA)
