This protocol describes direct Cy3 labelling of sheared DNA (as prepared by Total DNA isolation and shearing). This labelled DNA can be used as a normalizer in comparative expression experiments (hybridisations to cDNAs) or for strain diversity comparisons (DNA-DNA hybridisations). The protocol describes a modified application of the BioPrime array CGH labelling kit (Invitrogen 18095-012). For more detailed information concerning the kit check the manufacturer’s manual (Invitrogen 18095-013 and 18095-014; pdf document available in BASE). Ordering chemicals Cy3-dUTP solutions can be ordered at Amersham at 25 nmol scale (PA53022) or 250 nmol scale (PA53032). The concentration of the labelled nucleotide in both cases is 1 mM. For labelling DNA with Cy5 Cy5-dUTP is also available (PA55022) or 250 nmol scale (PA55032) NOTE: do not order labelled UTP’s (lacking the d), since they will not work, obviously!! Low-dTTP nucleotide mix Low-dTTp nucleotide mix (10x) 5 µl 100 mM dCTP 5 µl
Hybridization protocol
Two individual differentially, labeled cDNAs were, incubated at 95° C for 3', cooled down to 68° C and, mixed (final-volume 20 µl)., To these mixed cDNAs, 180 µl of pre-heated (68°, C) Slidehyb#1 hybridization, buffer (Ambion Austin USA), was added and the, resulting solution was, applied on a pre-heated, slide (68° C). Slides were, then hybridized at 44° C for, 16 hours. Subsequently, slides were washed at, 42°C once in 1 x, SSC/0.2% SDS and twice, in 1 x SSC and dried by, centrifugation (1 x SSC is, 0.15 M NaCl and 0.15 M, Sodium Citrate).
Scan protocol
Using the Scan Array, Express microarray, scanner. 1.Turn on the, scanner and the computer, (in this order) 2.Login:, scanner 3.Double click the, 'ScanArray Express' icon, 4.Switch on lasers 1 and 3, at least 15 minutes prior to, scanning arrays 5.Click the, 'File' button This is to, prevent you from, accidentally turning off the, laser after the 15 minutes, warming-up phase 6.Insert, the slide with the array-side, up and the label towards, the outside. On Agilent, slides the array side is the, side with the label that has, the word Agilent"""" on it, 7.Press 'Scan | Prescan', 8.Put resolution at 50 m, select both labels used, and set PMT values for, both channels 9.Press, 'Start' 10.Press 'Palette ', 'Green' as soon as, projection of the image has, started (first dye) select the, red colour as soon as, scanning of the second, layer (second dye) has, started 11.Adjust PMT, values to balance signals, obtained for both, channels. If necessary do, a few low-resolution scans, to obtain a optimal balance, 12.Click the 'Scan' button, again 13.Select frame for, high resolution scan adjust, resolution (to 10 m) and, scan the array for both, dyes 14.Press 'File | Save', to save the files 'Save all', saves both dye layers, Create a new folder for, each array 'Save all' saves, the signals from both layers, in individual files 15.Switch, off the lasers
Description
mouse on chow-3b
Data processing
After blank probe spots were removed, array measurements were first normalized using quantile normalization in R (http://www.r-project.org). Pearsons correlation on the quantile-normalized data was used to determine overall similarity between the culture medium and mouse-derived L. plantarum transcriptome data. The statistical significance of the differences was calculated from the variation in the biological replicates using Student's t-test on log(ratio) values. A FDR (false discovery rate) adjustment of the p-values was performed (Smyth, 2004). The list of differentially expressed genes were projected onto the metabolic pathways known for L. plantarum (Teusink et al., 2006) using the Simpheny software (Genomatica, Inc., San Diego, USA) and Cluster (Eisen et al, 1998) to display the gene expression patterns. Only genes with FDR adjusted p-values less than 0.05, corresponding to at most 5% false positives were displayed. Analysis was also assisted by LacPlantCyc (Teusink et al., 2005)
