RNA isolation and real time RT PCR for validation of microarray data Total RNA from laser captured AMC and RMC was extracted using miRNeasy Mini Kit and RNA from BV 2 cells was extracted with RNeasy Mini Kit according to the manufacturers instructions and quantified spectrophotometrically. 2 ug of RNA from each sample was added to a total towards volume of 25 ul reaction mixture contain ing 2. 5 uM of oligo primer, and 200U of Molony Murine Leukemia Virus Reverse Transcriptase. The reaction was initiated by incubating the reaction mixture for 1 h at 42 C for reverse transcription, and stopped by heating for 10 min at 70 C. Aliquot of the each reverse tran scription product was added to the 10 ul reaction mixture containing QuantiTectRSYBRR Green I, 0.

5 uM of each pri mer corresponding to Runx1t1, Sept9, Sept4, Mbp, Gapdh, Dcx, Mbp, or B actin Inhibitors,Modulators,Libraries and 4 mM MgCl2 to amplify the genes in ABi 7900HT Fast PCR system. The primer sequences of Runx1t1 are forward, and. The primer sequences used for the data reported in the supplementary Inhibitors,Modulators,Libraries figure are listed in Additional file 1, Sheet S1. After pre incubation at 95 C for 15 min, the polymerase chain reaction was performed as follows, 45 cycles of denaturation at 94 C for 15 s, annealing at 57 C for 25 s, and elongation at 72 C for 15 s. Results Laser capture microdissection of microglial cells from the corpus callosum of 5 day and 4 week old rat Inhibitors,Modulators,Libraries brain. To compare the gene expression profiles of AMC and RMC, we stained both microglial cell types with peroxidase conjugated lectin and isolated them from the CC of 5 day and 4 week old rat brain respectively.

LCM of Inhibitors,Modulators,Libraries AMC and RMC from the CC of 5 day old rat brain has been shown in Figure 1A F. Lectin staining has been widely used to selectively stain microglia for study of microglial development in the CNS. The cells iso lated by LCM were further confirmed to be microglia since the mRNA expression of oligodendrocyte, astrocyte and endothelial cell specific genes was undetectable. cDNA microarray and generation of gene lists specific to AMC and RMC To identify the genes that are differentially expressed be tween AMC and RMC, we extracted total RNA from AMC and RMC and carried out cDNA microarray using Rat Genome 230 2. 0 array. Each sample contained RNA from six hundred laser captured microglial cells.

To ensure gene expression consistency between samples within the groups, we determined the Pearsons rank correlation coefficient after normalizing the raw expression data. The gene expression profile from the samples of same group showed a Inhibitors,Modulators,Libraries very high correlation Erlotinib purchase of 0. 97 0. 03 while, a relatively lower correlation value of 0. 87 0. 03 was observed between samples of different groups. A high cor relation coefficient of above 0. 8 between the AMC and RMC may be due to the fact that the comparison is between the gene expression profiles of the same cell type, i. e. microglia regardless of the differences in age and morphology.