detailed growth protocol can be found in J. Anim Sci. 2008. 86:241-253.
Extracted molecule
total RNA
Extraction protocol
Total RNA from 1.0 g of tissue was extracted using TRIzol reagent (Invitrogen Corp., Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA concentration and quality were determined with an RNA 6000 Pico LabChip® kit using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Palo Alto, CA, USA).
Label
cy3
Label protocol
Five µg of total RNA were reverse transcribed with a T7 oligo(dT) primer using the Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion Inc., Austin, TX, USA) according to the manufacturer’s instructions. Following first-strand and second-strand synthesis and purification, the cDNAs were in vitro transcribed to synthesize multiple copies of amino allyl-modified aRNAs. After purification, aRNAs were labeled with N-hydroxysuccinate (NHS) ester Cy3 or Cy5 (GE Healthcare, UK).
detailed growth protocol can be found in J. Anim Sci. 2008. 86:241-253.
Extracted molecule
total RNA
Extraction protocol
Total RNA from 1.0 g of tissue was extracted using TRIzol reagent (Invitrogen Corp., Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA concentration and quality were determined with an RNA 6000 Pico LabChip® kit using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Palo Alto, CA, USA).
Label
cy5
Label protocol
Five µg of total RNA were reverse transcribed with a T7 oligo(dT) primer using the Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion Inc., Austin, TX, USA) according to the manufacturer’s instructions. Following first-strand and second-strand synthesis and purification, the cDNAs were in vitro transcribed to synthesize multiple copies of amino allyl-modified aRNAs. After purification, aRNAs were labeled with N-hydroxysuccinate (NHS) ester Cy3 or Cy5 (GE Healthcare, UK).
Hybridization protocol
Labeled aRNAs were purified and combined with 65 µl of Slide Hyb #1 solution (Ambion, Inc.) and denatured at 70C for 5 min. Hybridizations were performed in sealed hybridization cassettes (ArrayIt, TeleChem International, Inc., Sunnyvale, CA, USA) for 18 h at a humid 54C. Following hybridization, slides were washed in 2X SSC/0.5% SDS and 0.1X SSC/0.1% SDS solutions for 10 min each. The slides were rinsed in a 0.1X SSC solution and nuclease-free water and dried by centrifugation.
Scan protocol
Fluorescent images were detected by a GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA, USA) and fluorescence intensity data were collected using GenePix software (Molecular Devices) after spot alignment. Median intensity values for each dye channel were stored as comma-separated values data files.
Description
LD MUSCLE EXPRESSION PRP title indicates litter (number) and sex (G=female, B=male)
Data processing
Bioconductor package Marray was used for pre-processing. Values are log2 ratio Cy3/Cy5.
