|
| Status |
Public on Mar 31, 2015 |
| Title |
H37Rv in A549 #2 vs 7H9 replicate 2 (dye flip) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
7H9 broth grown M. tb
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: A549
|
| Treatment protocol |
none
|
| Growth protocol |
Monolayers of A549 cells in 4-5 T225 flasks were infected with M. tb H37Rv at an MOI of 5:1 bacteria to cell. Extracellular bacteria were removed by washing at 1 hr post-infection. Infected cells were maintained in 37C 5% CO2 for 72 hr pi then lysed and intracellular bacteria isolated. For reference, M. tb H37Rv was grown in Middlebrook 7H9 broth to log phase.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
M. tb H37Rv infected cells monolayer lysed with GTC solution and bacteria pelleted. Pellets were washed in GTC, resuspended in TRI reagent containing polyacryl carrier and disrupted in bead beater using sterile 0.1 mm zirconia. Total RNA was extracted according to published protocol (Dubnau E. et al. 2002). RNA from log phase culture of M. tb H37Rv grown in 7H9 broth was also extracted. An RNA amplification strategy was used (MessageAmp II-bacteria RNA amplification kit- Ambion) (Schlingemann et al., 2005; Garton et al., 2008).
|
| Label |
Cy3
|
| Label protocol |
The synthesis and fluorescent labeling of cDNA with Cy3 or Cy5 from amplified RNA (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 |
Mycobacterium tuberculosis H37Rv 72 hr in A549 Expt #2
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: A549
|
| Treatment protocol |
none
|
| Growth protocol |
Monolayers of A549 cells in 4-5 T225 flasks were infected with M. tb H37Rv at an MOI of 5:1 bacteria to cell. Extracellular bacteria were removed by washing at 1 hr post-infection. Infected cells were maintained in 37C 5% CO2 for 72 hr pi then lysed and intracellular bacteria isolated. For reference, M. tb H37Rv was grown in Middlebrook 7H9 broth to log phase.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
M. tb H37Rv infected cells monolayer lysed with GTC solution and bacteria pelleted. Pellets were washed in GTC, resuspended in TRI reagent containing polyacryl carrier and disrupted in bead beater using sterile 0.1 mm zirconia. Total RNA was extracted according to published protocol (Dubnau E. et al. 2002). RNA from log phase culture of M. tb H37Rv grown in 7H9 broth was also extracted. An RNA amplification strategy was used (MessageAmp II-bacteria RNA amplification kit- Ambion) (Schlingemann et al., 2005; Garton et al., 2008).
|
| Label |
Cy5
|
| Label protocol |
The synthesis and fluorescent labeling of cDNA with Cy3 or Cy5 from amplified RNA (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 grown in A549 cells was mixed with the labeled reference cDNA probe prior to purification with Microcon YM10 filter (Millipore). For each A549 grown M. tb sample (two biological replicates), the M. tb arrays were hybridized overnight with the mixed labeled cDNA probes in duplicate including a Cy3/Cy5 dye swap (Fontan et al, 2008). For biological replicate 1, 4 technical replicated including two dye flip were used.
|
| 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 WinPrint-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 |
Jun 13, 2014 |
| Last update date |
Mar 31, 2015 |
| Contact name |
Suman Laal |
| E-mail(s) |
[email protected]
|
| Organization name |
VA/NYU
|
| Department |
Pathology
|
| Lab |
Suman Laal
|
| Street address |
423 Esat 23rd Street, 18N
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10010 |
| Country |
USA |
| |
|
| Platform ID |
GPL4057 |
| Series (1) |
| GSE58466 |
Transcriptional Profile of Mycobacterium tuberculosis Replicating in Human Type II Alveolar Epithelial A549 cell line |
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