Quantitative real time polymerase chain reaction Messenger RNA expression level selleck chemicals Cisplatin for each HDAC was eval uated using an ABI 7500 machine. Amplifications were obtained using on demand TaqMan probes for each HDAC. For relative quantification of gene expression, standard curves were constructed for each gene by considering at least 3 points in triplicate of 10 fold serial dilution of cDNA in water, starting from 1 10 of a volume of undiluted cDNA transcribed from 1. 0 g of total RNA. The slopes of standard curves ranged from 3. 17 to 3. 87. Blank and standard controls were run in parallel to verify amplification efficiency within each experiment. To normalize differences in the amount of total cDNA added to each reaction, glucuro nidase gene expression was used as an endog enous control.
As a calibrator sample, the U343 cell line was used. To obtain the Ct values, we established a threshold of 0. 1. All reactions were made in duplicate, and all pro cedures were carried out at 4 C. Western Blotting For protein analysis, 30 g of each sample was loaded and separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Proteins were transferred to nitrocel lulose membranes, and the membranes were then incu bated in 1% Tris buffered saline Tween 20 containing 5% dried non fat milk for 1 hour at room temperature. The primary antibodies were diluted at 1 3000 in TBST containing 5% milk HDAC9, Acetyl Lys H3, and Acetyl Lys H4, and the membranes were incubated for 1 hour at room temperature, The membranes were then washed 3 times with TBST, incubated with the secondary antibody labeled by horseradish perox idase for 1 hour at room tem perature, and washed 3 times with TBST.
The secondary antibody was visualized using electron chemiluminescent reagent. Films were exposed from 10 to 60 seconds and developed. Statistical analysis Comparison of gene expression between groups of tumor was performed by nonparametric testes Mann Whitney and Kruskall Wallis. The level of significance was set at p 0. 05 in all analyses. Results Global expression of HDAC genes in gliomas and normal brain tissue The expression of 12 HDAC genes was analyzed using rel ative quantification of mRNA levels in normal brain, astrocytomas grades I, II and III, and glioblastomas. Class I HDAC genes showed lower levels of expression compared to the other classes studied.
The highest values of expression for class I were seen for HDAC8. Expression of HDAC class II and class IV were higher, with values of approximately 1. 0 to 150. 0. The highest level of mRNA was observed for HDAC9a and HDAC9b, with relative expression reaching values above 100 for normal brain tissue and grade I astrocytomas. however, HDAC6 and HDAC7 showed lev els of expression comparable with Entinostat those of class I. Table 1 shows the median values for each HDAC gene in all groups analyzed.