Array data processing and analysis was performed using Illu mina Bead Studio software. Hierarchical clustering ana lysis of significant genes was done using an algorithm of the JMP 6. 0. 0 software. Microarray analysis was per formed essentially as described. Raw microarray data were subjected to filtering and z normalization. Sample quality was assessed using scatterplots and gene sample z score based hierarchical clustering. Expression changes for individual genes were considered significant if they met 4 criteria, z ratio above 1. 4, false detection rate 0. 30, p value of the pairwise t test 0. 05, and mean back ground corrected signal intensity z score in each com parison group is not negative.
This approach provides a good selleck chemicals FR 180204 balance between sensitivity and specificity in the identification of differentially expressed genes, avoiding excessive representation of false positive and false nega tive regulation. All the microarray data are MIAME compliant and the raw data were deposited in Gene Expression Omnibus database. Real time reverse transcription quantitative PCR Total RNA was extracted with Trizol according to the manufacturers instructions. RNA was quantified and assessed using the RNA 6000 Nano Kit in the 2100 Bioanalyzer. One ug of total RNA from each cell line was used to generate cDNA using Taqman Reverse Transcription Reagents. The SYBR Green I assay and the GeneAmp 7300 Sequence Detection Sys tem were used for detecting real time PCR products. The PCR cycling conditions were as follows, 50 C, 2 min for AmpErase UNG incu bation, 95 C, 10 min for AmpliTaq Gold activation, and 40 cycles of melting and annealing exten sion.
PCR reactions for each template were performed in i thought about thisSofosbuvir PSI-7977 duplicate in 96 well plates. The com parative CT method was used to determine the relative expression in each sample using GAPDH as normalization control. The PCR pri mer sequences are available from the authors. Antibodies and Immunoblotting All the antibodies used for this work were obtained from commercial sources. Anti ABCB1 was purchased from GeneTex. Anti SPOCK2 and anti CCL26 were obtained from R D Systems. Anti PRSS8 and anti MSMB were obtained from Novus Biologicals. Anti GAPDH was purchased from Abcam. Immunoblotting was performed as previously described. Pathway Analysis We used WebGestalt version 2 to test for the enrichment of any pathway terms that may be related to the drug resis tance phenotypes. Two different databases were analyzed using Webgestalt. Overrepresenta tion analysis was also performed using the Reactome database. Ingenuity Pathway Analysis software was used to identify and draw net works relevant to the pathways identified.