6 protocols
nucleic_acid extraction
RNA was extracted using TriZol standard protocol
Microarray results are analyzed by GeneSpring GX 11 software (Agilent Technologies).Data transformation is applied to set a threshold at 1.0 of raw signals, followed by on-chip quantile normalization and subsequent transformation to base 2 logarithm.
Images at 5 um resolution are generated by Agilent scanner and the Feature Extraction 10.5 software is used to obtain the microarray raw-data.
Hybridizations are performed at 55°C for 17 hours in a rotating oven.
Labeled miRNAs is obtaino evidence of disease, according to Agilent's protocols, from 500 ng of total RNA through the ligation of a 5’-cytidine bisphosphate-Cy3 (pCp-Cy; Agilent Technologies) group at the 3’-end of each miRNA. To enhance the T4 RNA-ligase (Promega) efficiency, total RNA is previously treated with Alkaline Phosphatase (Amersham). Labeled miRNAs is purified by chromatography columns (Micro Biospin 6, Biorad).
treatment protocol
RNA was extracted from A375 cells, treated for 12 days with 20 uM of efavirenz or DMSO