strain background: Wistar tissue: left tibia sample group: Contralateral control
Extracted molecule
total RNA
Extraction protocol
The diaphysis of the tibia from experiment A was pulverized in presence of Trizol (Invitrogen) using the Freezer mill 6750 (Spex Certiprep, Metuchen, NY, USA) subsequently followed by a Trizol extraction, a chloroform/isoamylalcohol extraction, and a second Trizol extraction according to the manufacturer’s instructions. The RNA pellet was dissolved in RNase-free water and stored at 80ºC prior to use.
Label
Cy3
Label protocol
RNA from five rats of each group was used for microarray analysis cDNA probes were generated from 15 µg total RNA with an oligo-dT ((dT)20 VN) primer (Isogen, Maarssen, The Netherlands) and SuperScript™ II Reverse Transcriptase (Invitrogen), with incorporation of aminoallyl-dUTP (Ambion). Probes were indirectly labeled with Fluorolink Cy3 or Cy5 mono-functional dyes according to a dye-swap design. The dye-swap design was used to avoid a possible bias caused by the molecular structure of Cy3 and Cy5. The treated tibia (LOAD or SHAM) was hybridized together with its own contra lateral control tibia creating unique intra-rat hybridization.
strain background: Wistar tissue: right tibia sample group: LOAD (300 cycles (2 Hz) using a peak magnitude of 60 N)
Extracted molecule
total RNA
Extraction protocol
The diaphysis of the tibia from experiment A was pulverized in presence of Trizol (Invitrogen) using the Freezer mill 6750 (Spex Certiprep, Metuchen, NY, USA) subsequently followed by a Trizol extraction, a chloroform/isoamylalcohol extraction, and a second Trizol extraction according to the manufacturer’s instructions. The RNA pellet was dissolved in RNase-free water and stored at 80ºC prior to use.
Label
Cy5
Label protocol
RNA from five rats of each group was used for microarray analysis cDNA probes were generated from 15 µg total RNA with an oligo-dT ((dT)20 VN) primer (Isogen, Maarssen, The Netherlands) and SuperScript™ II Reverse Transcriptase (Invitrogen), with incorporation of aminoallyl-dUTP (Ambion). Probes were indirectly labeled with Fluorolink Cy3 or Cy5 mono-functional dyes according to a dye-swap design. The dye-swap design was used to avoid a possible bias caused by the molecular structure of Cy3 and Cy5. The treated tibia (LOAD or SHAM) was hybridized together with its own contra lateral control tibia creating unique intra-rat hybridization.
Hybridization protocol
The hybridization protocol was adapted from Snijders and colleagues [21] with minor modifications. Pre-hybridization was performed in a hybridization mixture containing 60 µg yeast tRNA (ribonucleic acid transfer, Sigma), 12 µg polyA (Amersham Biosciences) and 24 µg human Cot 1 DNA (Invitrogen) and 30 µg salmon sperm DNA (Invitrogen) in a total volume of 29.2 µl per array. After precipitation the pellet was dissolved in mastermix (50% formamide, 2xSSC, 9.4% dextran sulphate) and 0.2% SDS in a total volume of 130.2 µl per array. Pre-hybridization was maintained for 45 min at 37°C in the Hybridization Station (GenetacTM hybridization station or HybArray12 (Perkin Elmer)). The pre-hybridization mix was gently removed and slides were dried by centrifugation at 200 g for 3 min. The probe-hybridization mixture contained 16.0 µl Cy3 and 16.0 µl Cy5 labeled samples and 26.2 µl hybridization mixture (60 µg yeast tRNA, 12 µg polyA and 24 µg human Cot 1 DNA). After precipitation the pellet was dissolved in mastermix and 0.2% SDS in a total volume of 130.2 µl per array. Probe mix was denaturated at 70ºC for 15 min and incubated at 37ºC for 60 min. Hybridization was initiated and maintained for 16 h at 30ºC in the hybridization station, while the hybridization mixture was agitating. After hybridization, excess hybridization mixture was automatically rinsed off with 50% formamide, 2xSSC, pH 7.0 at 35ºC and followed by a wash step with PN buffer (0.1 M sodium phosphate, 0.1% Igepal CA630, pH 8) at 25ºC. Excess salt was removed by subsequently rinsing in 0.2xSSC, 0.1xSSC and 0.01xSSC at 25ºC. Slides were dried by centrifugation at 200 g for 3 min.
Scan protocol
After drying, the slides were scanned at 10 µm resolution for Cy3 and Cy5 intensities using the microarray scanner (Agilent Technologies) operated by Agilent Scan Control software and Feature Extraction software. Array images were processed with BlueFuse version 3.0 for microarrays (BlueGnome, Cambridge, UK).
Description
LOAD 2
Data processing
Automatically flagged genes (spots with a confidence value < 0.11) and manually flagged genes (dirty spots and spots with a confidence value between 0.11 0.15 with a low intensity or bad morphology) were excluded from further analysis. The duplicates of the Cy3 and Cy5 intensities were not averaged, but were treated as separate spots in the analysis. The log2 values of the Cy5 to Cy3 ratios were normalized in an intensity dependent fashion (lowess). Genes with more than one missing value across arrays were excluded from the statistical analysis. Differentially expressed genes were identified by fitting a separate linear model to the expression data for each gene using the language R (http://www.r-project.org). Duplicate spots for each gene, printed on each array, were taken into account [Smyth GK, Michaud J, Scott HS (2005), Bioinformatics. 21:2067-2075]. Differential expression was analyzed using moderated t statistics based on empirical Bayes estimation [Smyth GK, Michaud J, Scott HS (2005), Bioinformatics. 21:2067-2075] and P-values were adjusted according to the linear step-up method of Benjamini and Hochberg to reflect the false discovery rate [Benjamini Y, Hochberg Y (1995), Journal of the Royal Statistical Society Series B-Methodological 57:289-300; Reiner A, Yekutieli D, Benjamini Y (2003), Bioinformatics. 19:368-375]. Genes with a false discovery rate <20% were considered to reflect statistical significance. Estimated relative differential expression for LOAD (i.e., relative to its contra lateral control) was compared to the estimated relative differential expression for SHAM (i.e. relative to its contra lateral control). Thus, aspects of the experimental intervention bending and squeezing were compared with squeezing resulting in the net effect of bending.
