Muscles of the anterior and superficial posterior compartments were separately dissected, removed aseptically, pooled and finely minced with iris scissors to form a suspension which was seeded on multiple 100mm tissue culture dishes and left undisturbed overnight in an incubator at 37°C. Each plate contained 10 mls of growth media comprising Ham’s F10C (calcium=1.2mM, Sigma, St. Louis, MO), bovine basic fibroblast growth factor (bFGF: 6.6 ng/ml)(Sigma, St. Louis, MO), 200 ul chick embryo extract (CEE) (Invitrogen, Carlsbad, CA), and 10 ul fungizone (Gibco BRL, Gaithersburg MD). The muscle suspension was transferred to a 15ml conical tube on ice, triturated and vortexed vigorously for 30 seconds. The tubes were then centrifuged at low speed (Jouan BH-12: 1000 rpm x 5 min at 4C), the supernatant removed and the pellet resuspended in growth media for transfer to a 100mm dish. The plates were incubated overnight at 37°C and received supplemental feeding after 12 hours (66ng bFGF, 200ul CEE). The entire procedure beginning with trituration, was repeated each day for 5 days including preplating on plastic plates to remove fibroblasts. Eventually, a mix of purified myoblast clones remained that were seeded on denatured collagen (0.07% gelatin, Difco, Kansas City, MO) coated plates. All subsequent media exchanges (every 12 hours) involved complete growth media replacement but without chick embryo extract and these plates were expanded to a density of 0.8 x 106 cells/plate and the myoblasts were harvested for analysis.
Extracted molecule
total RNA
Extraction protocol
Frozen tissue specimens were transferred to 15 ml tubes (Sarstedt, Inc. Newton, NC) containing a ten-fold volume of TRIzol based on tissue weight (1ml/0.1mg; Life Technologies/Gibco, Gaithersburg,MD) and subjected to RNA purification as previously described (38). Homogenization was performed in these tubes followed by incubation at room temperature to permit complete dissociation of nucleoprotein complexes. Addition of 0.2 ml of chloroform per 1 ml of TRIzol reagent and centrifugation was performed to allow phase separation of the RNA. Total RNA was precipitated by mixing the aqueous phase with isopropanol. Samples were subsequently incubated, centrifuged and the supernatant removed. The pellet was washed with 75% ethanol, vortexed, centrifuged and air-dried followed by reconstitution in 100 ul of nuclease free water. For cultured cells, TRIzol was added directly to culture plates (5mls/100mm plate) after they were rinsed with ice cold PBS. The cells were harvested with a cell scraper into 15 ml tubes followed by homogenization. The aqueous layer was collected after organic phase separation as previously described and RNA was precipitated, washed with ethanol and resuspended in nuclease-free water as for the whole muscles. Criteria for inclusion in microarray studies was a spectrophotometric absorption ratio 260/280>1.8 (NanoDrop, Wilmington, DE) and RIN value >0.8 via electrophoretic analysis (Agilent Bioanalyzer 2100; Agilent Technologies, Santa Clara, CA).
Label
Biotin
Label protocol
In vitro transcription (IVT) of total RNA utilized the CodeLink™ Target Preparation Protocol (GE CodeLink™ Microarray System, Piscataway, NJ) as previously described (38). Briefly, 5 micrograms of purified RNA underwent cDNA synthesis using Invitrogen First and Second strand cDNA kits (Invitrogen, Austin,TX). Six bacterial control mRNAs were amplified in parallel to serve as fiducial markers and for intensity normalization during microarray scanning. Purification of double-stranded cDNA was performed using the QIAquick purification kit (Qiagen, Germantown, MD) and cRNA was synthesized via reverse transcriptase (Ambion, Austin, TX) in biotin-11-UTP (Perkin Elmer; Boston, MA) by incubation for 14 hours in a water bath at 37C. Biotin-labeled cRNA was recovered using an RNeasy Mini kit (Qiagen, Germantown, MD) and quantitation of the cRNA was performed by UV spectrophotometry at 260 nm. Confirmation of cRNA diversity was obtained using the Bioanalyzer 2100 to generate an electrophoretogram for each IVT reaction substrate which was characterized regarding sample yield, integrity and size diversity against a calibrated laboratory RNA standard. Based on evaluation of bacterial sequences incorporated as controls in the IVT assay, this step resulted in amplification of mRNA by only 50 to 100-fold thereby reducing the potential for errors associated with primer specific exponential amplification (77).
Hybridization protocol
CodeLink™ fragmentation buffer was added to the cRNA (5ul buffer/10ug cRNA) and incubated 20 minutes at 94C. Fragmented cRNA was suspended in hybridization solution containing nuclease-free water such that 250ul loading volume per array contained 10ug of cRNA. The solution was vortexed, heated (5 minutes at 90C) and transferred to ice prior to loading. Each slide contained a removable plastic hybridization chamber with an infusion port that was sealed immediately after loading. Microarrays were placed in a mixing incubator (Innova 4080 Shaking incubator, New Brunswick Scientific Co., Edison, NJ) for 18 hours, 300 rpm at 37C. CodeLink arrays were selected because of their high sensitivity and low error rate compared to other platforms especially regarding transcripts expressed at low copy numbers (73). After hybridization, arrays were individually taken from the incubator and their hybridization chambers were removed. The slides were placed in a reservoir containing 0.75X TNT (0.1M Tris-HCl, pH 7.6, 0.15 M NaCl, 0.05% Tween-20) for a 1 hour high-stringency wash at 46C. The arrays were incubated in Streptavidin Alexa Fluor 647 (Molecular Probes, Eugene, OR) for 30 minutes (0.2% Alexafluor-647 in TNB=0.1 M Tris-HCl, 0.15 M NaCl, 0.5% NEN Blocking Reagent, pH 7.6 (Perkin Elmer, Boston, MA). Slides were subsequently washed (1XTNT-5 min. each of 4 washes at RT; 0.1% SSC and 0.05% Tween20, 30 sec. at RT) and dried by low speed centrifugation.
Scan protocol
Slides were scanned using a 4000B GenePix scanner (Axon Instruments, Foster City, CA) that was calibrated prior to each use. Laser scanning parameters were set at 635 nm, PMT voltage at 600, resolution of 10 microns and the scan area adjusted for the entire array. Analysis of the array image was configured using the CodeLink™ Expression Analysis software (version 5.0). The program generates raw intensity outputs for each spot by segmentation and detection of spot intensity versus surrounding background. Data for each spot was determined as intensity per pixel within the probe zone after local background subtraction. The data for the array were then generated as both raw intensity values and normalized for the large dynamic range by dividing each spot by the overall median intensity value for that array. Values derived from poor or questionable spot profiles were removed from further computation as were all manufacturing errors designated by the CodeLink™ manufacturing spot report.
Description
Biotinylated cRNA probe is prepared and hybridization is carried out following the CodeLink protocols and reagents
Data processing
Raw intensities were normalized by CodeLink software based on global median for each array. CodeLink also calculated a threshold value based on the negative controls. The raw threshold value for this array is 8.058 and the normalized threshold value for this array is 0.263. To obtain the same data set we used for analysis, the normalized threshold value must be subtracted from each probe in the "value" column.
