|
| Status |
Public on Dec 31, 2015 |
| Title |
H37Ra 30oC_30h Vs 37oC_30h rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
H37Ra DnaAcos115_30C_30h
|
| Organism |
Mycobacterium tuberculosis H37Ra |
| Characteristics |
genotype/variation: DnaAcos115
culture condition: 30C for 30hrs
|
| Growth protocol |
Mycobacterium tuberculosis was grown in Middlebrook 7H9 broth supplemented with OADC and Kanamycin (10 mg/ml) at 30oC for 30h and shifted to 37oC for 30h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using protocol described (Fol et al. 2006; Mol. Micro. 60(3): 643)
|
| Label |
Cy3
|
| Label protocol |
Labeled c-RNA was prepared as described elsewhere (www.cag.icph.org). RNA was converted to double stranded cDNA using Random hexamers and Superscript (Invitrogen, CA). Briefly, 1.5µg total RNA was incubated with 5X first strand buffer, 0.1M DTT, 5mM each dATP, dGTP, dCTP and 0.2mM dTTP and 1.5 ul of Cy3 or Cy5 at 25oC for 10 min followed by 42oC for 90 min.
|
| |
|
| Channel 2 |
| Source name |
H37Ra DnaAcos115_37C_30hrs
|
| Organism |
Mycobacterium tuberculosis H37Ra |
| Characteristics |
genotype/variation: DnaAcos115
culture condition: 37C for 30hrs
|
| Growth protocol |
Mycobacterium tuberculosis was grown in Middlebrook 7H9 broth supplemented with OADC and Kanamycin (10 mg/ml) at 30oC for 30h and shifted to 37oC for 30h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using protocol described (Fol et al. 2006; Mol. Micro. 60(3): 643)
|
| Label |
Cy5
|
| Label protocol |
Labeled c-RNA was prepared as described elsewhere (www.cag.icph.org). RNA was converted to double stranded cDNA using Random hexamers and Superscript (Invitrogen, CA). Briefly, 1.5µg total RNA was incubated with 5X first strand buffer, 0.1M DTT, 5mM each dATP, dGTP, dCTP and 0.2mM dTTP and 1.5 ul of Cy3 or Cy5 at 25oC for 10 min followed by 42oC for 90 min.
|
| |
|
| |
| Hybridization protocol |
5µl of labeled sample in hybridization solution containing 0.5 µl of 10 mg/ml tRNA, 1.0 µl 20X SSC, 2.5 ul Formamide and 1.0 µl 1% SDS was heated to 98 oC for 2 minutes. The mix was Spun at 12,000 Xg for 2 min and allowed to cool for 5 min and then applied on to the microarray slide. Hybridization was carried out in standard hybridization chambers at 50oC for overnight.
|
| Scan protocol |
Images were quantified using GenePix4000B scanner (Molecular Devides)
|
| Description |
Comp17_30hr30C-S(cy3) vs 30hrPS-rep2(cy5)
|
| Data processing |
Images were processed using GenePix 5.1. Chips were normalized by the Print-tip Lowess method (Dudoit et al, 2000). Data were filtered by removing spots that were below the background noise. Spots were considered as below the background noise if the sum of the median intensities of the two channels is less than twice the highest mean background of the chiP.
|
| |
|
| Submission date |
Dec 24, 2013 |
| Last update date |
Dec 31, 2015 |
| Contact name |
Malini Rajagopalan |
| E-mail(s) |
[email protected]
|
| Phone |
903-877-7731
|
| Organization name |
University of Texas Health Sciences Center at Tyler
|
| Street address |
11937
|
| City |
TYLER |
| State/province |
Texas |
| ZIP/Postal code |
75708 |
| Country |
USA |
| |
|
| Platform ID |
GPL4057 |
| Series (1) |
| GSE53640 |
MtrA is involved in progression of Mycobacterium tuberculosis cell cycle |
|
