Sparc Tm1Cam knockout mouse, on a 129SvEv background, 9months old, male. Cortical femur, midsection, marrow flushed away before RNA extraction from bone. 129SvEv Sparctm1cam null mice: 129SvEv refers to mice of inbred strain 129 and substrain SvEv. Sparctm1cam refers to “Sparc, targeted mutation 1, University of Cambridge”. This targeted mutation is in exon 6 of the Sparc gene. Described in Gilmour et al, 1998 PMID: 9524110.
Biomaterial provider
Cardiff University, Dr. Fiona Mansergh.
Treatment protocol
Animals were sacrificed via cervical dislocation. Whole femurs and tibias were dissected free of surrounding tissue. Whole femurs were dissected free of surrounding tissue. Bone marrow was flushed via needle and syringe, using 0.5ml PBS per femur.A 0.4 cm section of the femoral midshaft was obtained in order to minimize contamination by muscle and cartilage adhering to femoral epiphyses and standardise anatomical localization. Material from 2-6 femoral cortices was pooled in RNAlater (Ambion, Huntingdon, Cambs, UK). Where tissue from more than one animal was used, material from litter mates was pooled.
Growth protocol
Mice were maintained and killed under Home Office licence in accordance with British law (comparable with U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals). Transgenic and control animals were maintained on RM3 diet (1.15% calcium, 0.82% phosphorus, 4088.65I.U./kg Vitamin D, Special Diet Services, Witham, Essex CM8 3AD, UK), ad libitum.
Extracted molecule
total RNA
Extraction protocol
Femoral midshaft samples were transferred from RNAlater into TRIzol (Invitrogen, Paisley, UK) and immediately homogenized using a Yellowline DI18 basic electronic homogenizer (Yellowline, IKA, Staufen, Germany) prior to RNA extraction via the manufacturer’s protocol. RNA samples were quantitated using formaldehyde gel electrophoresis and spectrophotometry (Camspec, Sawston, Cambs., UK).
Label
Cy3
Label protocol
Different pooled RNA samples were used for labelling array repetitions. 10ug of total RNA was labelled with either Cy3 or Cy5 dyes using the CyScribe labelling system (GE Healthcare, Chalfont St.Giles, Bucks, UK), according to the manufacturer’s protocol. 1ul of labelled cDNA was combined with 2ul 50% glycerol, run on a ‘John gel’ (a microscope slide sized, 1.5% agarose gel) and scanned using a GeneTac LS IV scanner (Genomic Solutions, Huntingdon, Cambs. UK) in order to assess successful incorporation of label. Control and experimental samples were then combined and prepared for hybridization.
129SvEv background wild type , 9months old, male. Cortical femur, midsection, marrow flushed away before RNA extraction from bone. Wild type inbred line from which the ES cells used to create the Sparc null mice were derived.
Biomaterial provider
Cardiff University, Dr. Fiona Mansergh
Treatment protocol
Animals were sacrificed via cervical dislocation. Whole femurs and tibias were dissected free of surrounding tissue. Whole femurs were dissected free of surrounding tissue. Bone marrow was flushed via needle and syringe, using 0.5ml PBS per femur.A 0.4 cm section of the femoral midshaft was obtained in order to minimize contamination by muscle and cartilage adhering to femoral epiphyses and standardise anatomical localization. Material from 2-6 femoral cortices was pooled in RNAlater (Ambion, Huntingdon, Cambs, UK). Where tissue from more than one animal was used, material from litter mates was pooled.
Growth protocol
Mice were maintained and killed under Home Office licence in accordance with British law (comparable with U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals). Transgenic and control animals were maintained on RM3 diet (1.15% calcium, 0.82% phosphorus, 4088.65I.U./kg Vitamin D, Special Diet Services, Witham, Essex CM8 3AD, UK), ad libitum.
Extracted molecule
total RNA
Extraction protocol
Femoral midshaft samples were transferred from RNAlater into TRIzol (Invitrogen, Paisley, UK) and immediately homogenized using a Yellowline DI18 basic electronic homogenizer (Yellowline, IKA, Staufen, Germany) prior to RNA extraction via the manufacturer’s protocol. RNA samples were quantitated using formaldehyde gel electrophoresis and spectrophotometry (Camspec, Sawston, Cambs., UK).
Label
Cy5
Label protocol
Different pooled RNA samples were used for labelling array repetitions. 10ug of total RNA was labelled with either Cy3 or Cy5 dyes using the CyScribe labelling system (GE Healthcare, Chalfont St.Giles, Bucks, UK), according to the manufacturer’s protocol. 1ul of labelled cDNA was combined with 2ul 50% glycerol, run on a ‘John gel’ (a microscope slide sized, 1.5% agarose gel) and scanned using a GeneTac LS IV scanner (Genomic Solutions, Huntingdon, Cambs. UK) in order to assess successful incorporation of label. Control and experimental samples were then combined and prepared for hybridization.
Hybridization protocol
Array slides were incubated in prehybe for 1 hour at 42oC (50% formamide, 5XSSC, 0.1% SDS, 1% BSA). Targets were dried down via vacuum centrifugation then resuspended in 50l hybe solution (49.9% de-ionised formamide, 49.9% 20xSSC, 0.2% SDS) with added 1l Cot1 DNA and 1l poly A oligo as blocking agents, heated to 95oC for 5 minutes and then added to the face of one slide. The printed face of the second slide of the pair was then placed face to face with the first, using the same probe. Slide pairs were then placed on a level plastic cover above some 1xSSC moistened tissue in a slide box. The slide box was sealed with Nescofilm (Karlan Research Products Corporation, Santa Rosa, CA, USA), placed floating in a waterbath and hybridized for 24-48 hours at 42oC. Following hybridization, slides were washed once in Wash solution 1 (1X SSC, 2% SDS, filtered autoclaved ddH2O) for 20 minutes, then twice in Wash solution 2 (0.1X SSC, 0.2% SDS, filtered autoclaved distilled deionised H2O (ddH2O) for 20 minutes each. Slides were dipped in nuclease free filtered water, then spray dried, finally, the backs of the slides were cleaned with filtered autoclaved ddH2O, then wiped with 100% EtOH, then wiped dry and scanned.
Scan protocol
Scans were carried out at 12.5 micrometers, using the averaging setting (GeneTac LSIV scanner, (Genomic Solutions, Cambridgeshire, UK)). It is possible to carry out quick draft scans using this scanner, gain and black settings, which affect image intensity and background were varied slightly in order to optimize the signal/noise ratio for each channel and each slide before proper scans were initiated.
Description
Arrays were repeated 5 times with fluor switching, in order to counteract any issues of dye bias that may have arisen from direct labelling. Repetition sets used biological samples derived from at least four control and four experimental animals per set, in order to control for biological variation between individual animals. Scanned images were stored and filtered, then analysed using the GeneTac Analyser spot finding software (Genomic Solutions, Cambridgeshire, UK). Analysis and other methods were carried out similarly to another published experiment, please see Mansergh et al. 2004 PMID: 15303089 for more details.
Data processing
Output files from GeneTac analyser were saved in MS Excel spreadsheet format. MS Excel was used for all further data manipulation. We normalised each channel via total array methods, via calculation of the mean intensity value. Normalised intensities were then analysed using two different methods. Firstly, we transferred normalised intensity values for each of 5 experimental repetitions into a separate Excel file, ensuring a standard order of samples. These data were then formatted for Significance Analysis of Microarrays (SAM; http://www-stat.stanford.edu/~tibs/SAM ). SAM analysis was carried out and genes that showed a fold change of 2 or above and that appeared in a SAM analysis as statistically significant above a delta value of 0.5 (which denotes an error rate of 5%) were selected for further appraisal. SAM, however, has some disadvantages; all replicates have to be in the same order to use SAM, precluding some methods of data filtration. In addition, each cDNA probe (represented by a single accession number) is spotted on the HGMP NIA array slides twice. Using SAM, it is impossible to assess both duplicate spots together. Hence, we supplemented SAM analysis with a second analysis method as follows: Following normalisation, we used approximately 700 blank spots per slide to calculate a mean background value + 2 standard deviations of that background value, for each channel. Genes that fell below this cutoff in BOTH control and experimental channels were removed. Genes that failed to show a fold change of 2 were also removed. Remaining data (above background + 2SD values, fold change of > 2) were retained, combined with similar data from other replicates and sorted via accession number. Genes that appeared above background + 2SD values with a fold change of > 2, in at least 8 out of 10 replicates (this analysis combined values from duplicate spots, giving 10 replicates per EST), were retained for further analysis. Finally, accession numbers obtained from this method and from SAM were compared. Genes appearing as differentially regulated using BOTH methods were deemed significant by us. In other words, significant ESTs are changed in expression by at least two fold, are above background + 2SD in at least one channel and have a delta value of 0.5 using SAM. These ESTs were subjected to bioinformatic analysis and were ALL assessed via RT PCR in order to confirm array results.
