|
| Status |
Public on Apr 18, 2017 |
| Title |
sputum from pulmonary tuberculosis patient 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
7H9 broth-grown M. tb
|
| Organism |
Mycobacterium tuberculosis |
| Characteristics |
bacterial strain: H37Rv
human tissue source: none (broth-grown)
|
| Treatment protocol |
Expectorated sputum were immediately transferred to Primestore molecular transport media after collection.
|
| Growth protocol |
For using as reference, M. tb H37Rv was grown in Middlebrook 7H9 broth to mid-log phase
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Sputum samples after collection were immediately transferred to Primestore molecular transport media (Longhorn vaccines and diagnostics). Total RNA from sputum samples in primestore was isolated using RNAzol as per manufacturer's protocol. An RNA amplification strategy was used (MessageAmp II-bacteria RNA amplification kit- Ambion) (Schlingemann etal., 2005; Garton et al., 2008)
|
| Label |
Cy3
|
| Label protocol |
The synthesis and fluorescent labeling of cDNA with Cy3 or Cy5 from aRNA was accomplished by first performing reverse transcriptase (RT) reaction with Superscript II RT followed by labeling with Klenow fragment using Bioprime Kit (Invitrogen) following a published method (Schlingemann 2005).
|
| |
|
| Channel 2 |
| Source name |
sputum from PTB patient 3
|
| Organism |
Mycobacterium tuberculosis |
| Characteristics |
bacterial strain: speciation not done
human tissue source: sputum
diagnosis: tuberculosis
|
| Treatment protocol |
Expectorated sputum were immediately transferred to Primestore molecular transport media after collection.
|
| Growth protocol |
For using as reference, M. tb H37Rv was grown in Middlebrook 7H9 broth to mid-log phase
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Sputum samples after collection were immediately transferred to Primestore molecular transport media (Longhorn vaccines and diagnostics). Total RNA from sputum samples in primestore was isolated using RNAzol as per manufacturer's protocol. An RNA amplification strategy was used (MessageAmp II-bacteria RNA amplification kit- Ambion) (Schlingemann etal., 2005; Garton et al., 2008)
|
| Label |
Cy5
|
| Label protocol |
The synthesis and fluorescent labeling of cDNA with Cy3 or Cy5 from aRNA was accomplished by first performing reverse transcriptase (RT) reaction with Superscript II RT followed by labeling with Klenow fragment using Bioprime Kit (Invitrogen) following a published method (Schlingemann 2005).
|
| |
|
| |
| Hybridization protocol |
For hybridization, labeled cDNA probes generated from aRNA obtained from M. tb in PTB patient sputum was mixed with the labeled reference cDNA probe prior to purification with Microcon YM10 filter (Millipore). For each sputum sample from PTB patients, the M. tb arrays were hybridized over night with the mixed labeled cDNA probes including a Cy3/Cy5 dye swap for 4/7 samples (Fontan et al, 2008)
|
| Scan protocol |
The microarrays were scanned and processed with an Axon 4000B scanner and GenePix Pro 6.1 software, respectively.
|
| Data processing |
Normalized by the print-tip Lowess method; intensity ratio data obtained used to perform Significance Analysis of Microarrays (SAM) with Multiarray Viewer Software on the TMEV website for determination of differentially expressed genes; ≥ 2 fold change at a false discovery rate of < 2% were considered significantly differentially expressed.
|
| |
|
| Submission date |
Jan 09, 2017 |
| Last update date |
Apr 18, 2017 |
| Contact name |
Indu Verma |
| E-mail(s) |
[email protected]
|
| Phone |
09815177700
|
| Organization name |
Post Graduate Institute of Medical Education and Research (PGIMER)
|
| Department |
Biochemistry
|
| Lab |
310
|
| Street address |
Dept. of Biochemistry, PGIMER
|
| City |
Chandigarh |
| State/province |
Outside U.S. & Canada |
| ZIP/Postal code |
160012 |
| Country |
India |
| |
|
| Platform ID |
GPL4057 |
| Series (1) |
| GSE93316 |
Transcriptome analysis of mycobacteria in sputum samples of pulmonary tuberculosis patients |
|
