|
| Status |
Public on Dec 31, 2014 |
| Title |
INH 48 hrs |
| Sample type |
RNA |
| |
|
| Source name |
INH 48 hrs
|
| Organism |
Mycobacterium tuberculosis H37Rv |
| Characteristics |
strain: H37Rv
treatment: Cells treated with isoniazid
time: 48hr
|
| Treatment protocol |
M. tuberculosis H37Rv cells were grown to an OD580 of 0.6 in Middlebrooks 7H9 broth (Difco BBL) containing 0.5% (v/v) glycerol, 0.05% (v/v) Tween-80 and supplemented with 10% (v/v) OADC.The culture was aliquoted to sterile screw cap tubes. EPMC was added to make a final concentration of 2.42 mM (5 X MIC). Isoniazid at 1.45 µM served as a positive control. Untreated control consisted of the bacterial culture treated with no drug but only with dimethyl formamide (DMF) which was used to dissolve EPMC.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The isolation of RNA was carried out using RNAspin mini Kit (GE Lifesciences) as per the protocol provided by the manufacturer. Finally the RNA was eluted in 60 µl of RNase-free water and stored frozen at -80 °C until use. Total RNA purity was assessed by the NanoDrop® ND-1000 UV-Vis Spectrophotometer (Nanodrop technologies, Rockland, USA).
|
| Label |
Cy3
|
| Label protocol |
The RNA samples were labeled using Quick Amp Kit PLUS (Agilent, Palo Alto, CA) using cy3 CTP.
|
| |
|
| Hybridization protocol |
The labeled cRNA samples were hybridized onto Agilent Custom Mycobacterium tuberculosis 8x15k slide format (Genotypic Technology Pvt. Ltd, Bangalore). Six hundred ng of Cy3 labeled samples were fragmented and hybridized. Fragmentation of labeled cRNA and hybridization were done using the Gene Expression Hybridization kit (Agilent). Hybridization was carried out in Agilent’s Surehyb Chambers at 65 ºC for 16 hours. The hybridized slides were washed using Agilent Gene Expression wash buffers and scanned using the Agilent Microarray Scanner at 5 micron resolution.
|
| Scan protocol |
Slide was scanned using Agilent Scanner
|
| Description |
Cells treated with isoniazid for 48 hrs
US83000164_251994310013_S01_GE1_105_Dec08_1_4
|
| Data processing |
Feature extracted data was analyzed using GeneSpring GX version 10 software (Agilent, Palo Alto, CA). (Gene based Analysis) Normalization of the data was done in GeneSpring GX using the 75th percentile shift and Normalization to Specific Samples. Genes showing upregulation and downregulation by one fold and above among the test samples compared to untreated control samples were identified. Differentially regulated genes were clustered using hierarchical clustering based on Pearson coefficient correlation algorithm to identify significant gene expression patterns.
|
| |
|
| Submission date |
Oct 15, 2012 |
| Last update date |
Dec 31, 2014 |
| Contact name |
Ajay Kumar |
| E-mail(s) |
rakumar@rgcb.res.in
|
| Organization name |
Rajiv Gandhi Centre for Biotechnology
|
| Department |
Molecular Microbiology
|
| Lab |
Mycobacterium Research Group
|
| Street address |
Thycaud P.O.
|
| City |
Trivandrum |
| State/province |
Kerala |
| ZIP/Postal code |
695014 |
| Country |
India |
| |
|
| Platform ID |
GPL16177 |
| Series (1) |
| GSE41582 |
Analysis of global transcriptional response of Mycobacterium tuberculosis H37Rv to the natural compound ethyl p-methoxycinnamate |
|
