|
| Status |
Public on Mar 31, 2014 |
| Title |
HIV+ blood donor 4, replicate 2 (dye flip) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
M. tb in HIV+ blood (donor H28)
|
| Organism |
Mycobacterium tuberculosis H37Rv |
| Characteristics |
donor: H28
hiv status: HIV+
growth protocol: M. tb in HIV+ blood
|
| Growth protocol |
Fresh human whole blood from 6 HIV- and 6 HIV+ was diluted with equal volumes RPMI-1640 supplemented with L-glutamine and heparin and infected with M. tb H37Rv (10^6 CFU/ml) and incubated at 37°C shaking at 20 rpm for 96 hr. Fot reference, M. tb H37Rv was grown in Middlebrook 7H9 broth to log phase.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Blood cells lysed with 0.1% triton X 100 with 6M urea and bacteria and cells pelleted. Pellets imediately resuspended in GTC solution for lysis of remainig cells. After washing with GTC, bacterial pellets suspended in TRI reagent with polyacrylamide carrier and disrupted in bead beater using sterile 0.1 mm zirconia. Total RNA was extracted accordig to published protocol (Dunau E. e al. 2002). 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 |
7H9 broth-grown M. tb (reference)
|
| Organism |
Mycobacterium tuberculosis H37Rv |
| Characteristics |
growth protocol: 7H9 broth-grown
|
| Growth protocol |
Fresh human whole blood from 6 HIV- and 6 HIV+ was diluted with equal volumes RPMI-1640 supplemented with L-glutamine and heparin and infected with M. tb H37Rv (10^6 CFU/ml) and incubated at 37°C shaking at 20 rpm for 96 hr. Fot reference, M. tb H37Rv was grown in Middlebrook 7H9 broth to log phase.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Blood cells lysed with 0.1% triton X 100 with 6M urea and bacteria and cells pelleted. Pellets imediately resuspended in GTC solution for lysis of remainig cells. After washing with GTC, bacterial pellets suspended in TRI reagent with polyacrylamide carrier and disrupted in bead beater using sterile 0.1 mm zirconia. Total RNA was extracted accordig to published protocol (Dunau E. e al. 2002). 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 grown in each blood sample was mixed with the labeled reference cDNA probe prior to purification with Microcon YM10 filter (Millipore). For each M. tb-infected blood sample, the M. tb arrays were hybridized over night with the mixed labeled cDNA probes in duplicate including a Cy3/Cy5 dye swap (Fontan et al, 2008)
|
| Scan protocol |
The microarrays were scanned and processed with an Axon 4000B scanner and GenePix Pro 6.1 software, respectively.
|
| Description |
Sample name: h28cy3RVcy5
total RNA (amplified)
|
| 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 |
Aug 11, 2013 |
| Last update date |
Mar 31, 2014 |
| 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) |
| GSE49760 |
Transcriptional profiling of Mycobacterium tuberculosis H37Rv in whole blood from HIV- and HIV+ individuals |
|
