Title: Affymetrix CEL analysis. Description:
Title: Affymetrix Generic Hybridization. Description:
RNA was isolated using the RNeasy Plant Mini Protocol (QIAGEN, Clifton Hill, Australia) and the quality of the RNA was assessed using the Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA) where spectrophotometric analysis was carried out to determine the A260 to A280 ratio and the concentration. For qRT-PCR, cDNA synthesis was carried out using the Biorad (Perth) iScript cDNA synthesis kit according to manufacturers instructions. For arrays, the cRNA was prepared from 3 ug of total RNA almost exactly as described in the Affymetrix GeneChip Expression Analysis Technical Manual.
Following sample hybridisation and scanning of the individual arrays, Affymetrix Expression Console v1.1.2 was used to assess the overall quality control of each batch within the array experiment and overall using Robust multichip average (RMA) normalisation (Bolstad et al., 2003; Irizarry et al., 2003) and Affymetrix default chip annotations . The main quality control indices examined were the bacterial spike in controls for the hybridisation stage and the Poly-A controls used in both the Affymetrix WT cDNA synthesis and Amplification kit and the Ambion WT Expression Kit to assess for uniformity of these controls as well as the overall mean probe signal and background signal as well as the overall signal histogram. Probes defined on the Rat Gene 1.0 ST chip were filtered for the presence of the 2,520,602 single-nucleotide polymorphisms and 689,940 insertions and deletions present between the WKY and LEW genomes derived from whole genome sequencing of the strains on the Illumina HiSeq platform (Mr Santosh Atanur, Physiological Genomics and Medicine Group, MRC Clinical Sciences Centre, Imperial College, personal communication and unpublished data) through the creation of a custom chip definition file (CDF) which also reflected the up to date information from current genome and transcriptome databases to give greater accuracy of the probe set definitions than the standard Affymetrix CDF files (Dai et al., 2005). The SNP filtering reduced the number of probes from 8,460,613 to 8,250,260, a reduction of 2.5% in total. RMA normalisation (Quantiles) was performed on the sample set and probe sets were removed that showed that a transcript was not expressed in the cell using a signal threshold of 100 (log2 value = 6.64). Probe sets were removed if this threshold was not passed in at least one sample from the whole series of strains and time points for that probe set.
RNA was isolated from homogenised cultured BMDMs using TRIzol reagent (Invitrogen). To each 6cm petri or well of a 6 well culture plate, 1ml TRIzol reagent was added and TRIzol-lysed cell solution stored at -80C prior to RNA extraction. (Parameters: Extracted product = total_RNA, Amplification = none)
BMDMs were prepared from the femurs of parental and congenic strains using previously described methods (Behmoaras et al., 2008) with some key modifications. For all applications, ‘full culture’ medium was used to differentiate bone marrow stem cells into BMDMs and consisted of DMEM and 2mM L-glutamine (Gibco, Invitrogen Ltd, Paisley, UK) and 25mM Hepes buffer (Sigma-Aldrich, Gillingham, UK), 25% L929 conditioned media, 25% decomplemented foetal bovine serum (F-539, M.B. Meldrum, Bourne End, UK), penicillin (100U/ml, Invitrogen) and streptomycin (100ug/ml, Invitrogen). Femurs were isolated from adult male rats aged 8-10 weeks of age. The femurs were stripped of all muscle tissue and washed twice in 100% ethanol. The ends of the femurs were cut off with bone scissors and 10ml of cold Hanks buffer flushed through each end of the femur into a 50ml falcon tube using a 20 gauge needle to extract the bone marrow. The samples were centrifuged at 40C for 5 minutes at 1500rpm and the supernatant discarded. The resulting cell pellets were resuspended in 10ml Hanks and the 50ml tubes incubated for 15 minutes in a 5% CO2 incubator to allow for the lysis of the red cells to occur. The cells were centrifuged at 40C for 5 minutes at 1500 rpm and the supernatant discarded prior to resuspension of the cell pellet in 1ml of full culture media. The cells were dispensed equally (250ul of resuspension) amongst 4 large (15cm) petri dishes (Sterilin, Newport, UK) prefilled with 30ml of full culture media for chromatin immunoprecipitation (ChIP) and siRNA knockdown experiments and 50ul of cell resuspension in 6cm petri dishes (Nunc, Roskilde, Denmark) filled with 5ml full culture media for gene expression experiments. BMDMs were cultured for 4 days in a 5% CO2 incubator at 37C. The resulting cells were characterised as macrophages by ED-1 (CD68) immunohistochemistry (Parameters: start time = 4, time unit = days, min temperature = 37, temperature unit = C, media = 25% L929 conditioned media)
On day 5 of BMDM culture, petri dishes were washed with cold Hanks solution and culture medium changed to fresh full culture medium supplemented with 100ng/ml LPS (lipopolysaccharides from Salmonella typhosa purified by gel-filtration chromatography, Sigma-Aldrich) for chromatin immunoprecipitation and gene expression studies. BMDMs were incubated for the desired time and then processed accordingly for the appropriate experiment.
After following the Extraction and differentiation of BMDMs protocol. On day 4 the BMDMs were harvested and replated in 6 well culture plates (Nunc). Briefly, the 15cm petri dish was washed with 10ml cold Hank’s balanced salt solution (HBSS, Gibco, Invitrogen Ltd, Paisley, UK) then 12ml of non-enzymatic cell dissociation solution (Sigma-Aldrich) was added and the petri dish placed in the incubator for 30 minutes. The dish was removed and 5ml of full culture media added to the petri dish to stop the action of the dissociation solution and the plate washed to dislodge the cells. The resulting cell suspension was transferred to a 50ml falcon tube and kept on ice until all cells were recovered from the remaining petri dishes. The cells were centrifuged at 1500rpm for 5 minutes at 4C. The supernatant was discarded and the cells resuspended in 1ml of complete media. 2ml of pre warmed full culture media was added into each well of the 6 well culture plates (Nunc). The cell concentration was counted using a haemocytometer and 1 million cells dispensed into each well. The plate was rocked gently back and forth to ensure even cell distribution. (Parameters: start time = 5, time unit = days, min temperature = 4, max temperature = 37, temperature unit = C)
Transfections with siRNA were commenced on day 6 of culture. To prepare the transfection mixture, 2.64 ml Optimem (Invitrogen) was incubated with 60ul Dharmafect 1 (Dharmacon) for 5 minutes at room temperature. At the same time 240ul of Optimem was incubated with 60ul siRNA (20uM) for 5 minutes at room temperature. The two preparations were then mixed and incubated at room temperature for 20 minutes with frequent mixing by tube inversion to allow the integration of siRNA particles into the liposomal particles contained within Dharmafect transfection reagent. Meanwhile the media in the 6 well culture plates was removed, the cells washed with DMEM and then 1.5ml DMEM only with no supplementation was added to each well as the presence of serum containing medium inhibits adequate transfection with siRNA (J. Behmoaras, Centre for Complement and Inflammation Research, Imperial College, personal communication). Five hundred microlitres of the transfection mixture was added to each well and mixed resulting in a final siRNA concentration of 100nM. The cells were incubated for 48 hours in a 5% CO2 incubator at 37C. On day 8 the cells were stimulated with LPS or kept in the basal state and the supernatants collected and stored at -80C and the cells homogenised using 1ml TRIzol reagent for future RNA extraction