|
| Status |
Public on Dec 08, 2015 |
| Title |
ST701-LMn vs ST701-HMn_rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
ST70 (mntR mutant of Mycobacterium tuberculosis)
|
| Organism |
Mycobacterium tuberculosis |
| Characteristics |
strain: ST70
treatment: Low manganese
|
| Treatment protocol |
Cells were grown in the minimal media supplemented with 50uM manganese or without manganese
|
| Growth protocol |
Rv (Mycobacterium tuberculosis wild type strain) and ST70 (mntR mutant strain of Mycobacterium tuberculosis) were grown, as three independent biological replicates, in minimal media without manganese or with 50uM manganese upto optical density 0.2 and then the cells were harvested
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
Synthesis and Labeling of cDNA 1. Bring 1.5µg of RNA to a volume of 11µl (in a PCR tube) with nucleotide-free water and add 2.2µl (2mg/ml) random primers (pulse spin to collect contents). 2. Heat 2 min at 98oC, snap cool on ice. 3. (Keep on ice) and add 11.1µl of the following reaction mix: 5.0µl 5X First-Strand buffer 2.5µl 100 mM DTT 2.3µl low dTdNTP mix (5mM A,G,C and 0.2mM T stock) 1.5ul Cy3 or Cy5 Then add 1.2ul Superscript, vortex to mix and spin to collect contents 4. Incubate 10 min at 25oC followed by 90 min at 42oC in PCR machine. **Can freeze or leave at 4oC overnight if necessary 5. Prime an Amicon Ultra 10K column with 400µl of 25% TE and spin for 5 min at 14,000 Xg. Empty flow-through from collection tube and return primed column. Add another 400ul of 25% TE followed by both reactions (cy3 and cy5) to column and centrifuge at 14,000 Xg for 7 min. Empty flow-through, and add yet another 400ul of 25% TE, centrifuge at 14,000 Xg for 7 min. Empty flow-through once more, add 400ul of 25% TE and centrifuge at 14,000 Xg for 12 min. Empty the flow-through. 6. Recover cDNA by inverting Amicon filter into the same collection tube and centrifuge at 1,000 Xg for 2 min. 7. Using a speedvac, concentrate sample to 5ul. The detailed labeling and hybridization protocol can be obtained at http://www.cag.icph.org/downloads_page.htm#LabelingProtocols.
|
| |
|
| Channel 2 |
| Source name |
ST70 (mntR mutant of Mycobacterium tuberculosis)
|
| Organism |
Mycobacterium tuberculosis |
| Characteristics |
strain: ST70
treatment: 50uM MnCl2 manganese
|
| Treatment protocol |
Cells were grown in the minimal media supplemented with 50uM manganese or without manganese
|
| Growth protocol |
Rv (Mycobacterium tuberculosis wild type strain) and ST70 (mntR mutant strain of Mycobacterium tuberculosis) were grown, as three independent biological replicates, in minimal media without manganese or with 50uM manganese upto optical density 0.2 and then the cells were harvested
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
Synthesis and Labeling of cDNA 1. Bring 1.5µg of RNA to a volume of 11µl (in a PCR tube) with nucleotide-free water and add 2.2µl (2mg/ml) random primers (pulse spin to collect contents). 2. Heat 2 min at 98oC, snap cool on ice. 3. (Keep on ice) and add 11.1µl of the following reaction mix: 5.0µl 5X First-Strand buffer 2.5µl 100 mM DTT 2.3µl low dTdNTP mix (5mM A,G,C and 0.2mM T stock) 1.5ul Cy3 or Cy5 Then add 1.2ul Superscript, vortex to mix and spin to collect contents 4. Incubate 10 min at 25oC followed by 90 min at 42oC in PCR machine. **Can freeze or leave at 4oC overnight if necessary 5. Prime an Amicon Ultra 10K column with 400µl of 25% TE and spin for 5 min at 14,000 Xg. Empty flow-through from collection tube and return primed column. Add another 400ul of 25% TE followed by both reactions (cy3 and cy5) to column and centrifuge at 14,000 Xg for 7 min. Empty flow-through, and add yet another 400ul of 25% TE, centrifuge at 14,000 Xg for 7 min. Empty flow-through once more, add 400ul of 25% TE and centrifuge at 14,000 Xg for 12 min. Empty the flow-through. 6. Recover cDNA by inverting Amicon filter into the same collection tube and centrifuge at 1,000 Xg for 2 min. 7. Using a speedvac, concentrate sample to 5ul. The detailed labeling and hybridization protocol can be obtained at http://www.cag.icph.org/downloads_page.htm#LabelingProtocols.
|
| |
|
| |
| Hybridization protocol |
Prehybridization 1. Prepare Prehybridization Solution (Fresh Each Time) **this volume is based on a glass dish with a base measurement of 11x8cm. This dish allows for a pre-hyb of 1-4 slides. Adjust volume accordingly based on size of dish and/or number of slides. 106.0ml dH2O 3.0g BSA 1.2ml 10%SDS 2. Add Prehybridization solution to a glass slide-staining dish and place post-processed slides array side up into solution. Make sure there are no bubbles on the surfaces of the slides. 3. Place the staining jar containing the Prehybridization solution and slides into a waterbath and hybridize for 1 hour at 42oC. 4. Take the prehybridized slides out of the Prehybridization solution and place into a slide holder submerged in a slide-washing tray containing nuclease-free water. Wash slides for 2 minutes with constant shaking up and down. 5. Transfer slide rack to a slide washing tray containing isopropanol and wash for 2 minutes with constant shaking up and down. 6. Take the slides in the isopropanol bath to the centrifuge to keep the slides wet until you can dry them rapidly. Spin at 600 RPM in a 50ml conical for 3 minutes to dry. **Complete this step within one hour of hybridization III. Hybridization 1. Add the following to the 5µl labeled sample. 0.5µl 10 mg/ml tRNA 1.0µl 20X SSC 2.5ul Formamide 1.0µl 1% SDS 2. Heat to 98 oC for 2 minutes. Spin in centrifuge for 1 min at 12,000 Xg. Let cool for 5 min. 3. Pipette cooled 10µl hybridization solution on microarray. Apply 22 x 22 coverslip to cover array (if bubbles appear under the cover slip, they will slowly migrate to the edges provided that the volume is accurate). 4. OPTIONAL: Apply thin wall of rubber cement around the 4 corners of the coverslip to avoid movement. This step is recommended if your waterbath is not perfectly level. 5. Hybridize in standard hyb. chambers with ~40µl water under each slide (20ul under both ends of each slide) overnight in 50oC water bath **Bubbles (esp. large ones) can be troublesome for your arrays since they can cause areas of non-hybridization. To avoid bubbles, do not use the blowout feature of the pipette when adding the probe to the slide. Also, clean your coverslips with alcohol and lint-free wipes before placing over probe. IV. Post Hyb washes (next day) 1. Remove cover slip while array is submerged in first wash solution (2XSSC + 0.1% SDS), transfer to submerged microscope slide staining racks and rinse for 1 min in 1X SSC+0.05% SDS with agitation/shaking. 2. Rinse in 0.06X SSC with shaking. Transfer to another submerged staining rack and wash 2 min in fresh 0.06X SSC with shaking. 3. Centrifuge 3 min at 1000 rpm in a 50ml conical (array edge up) to dry. Scan immediately.
|
| Scan protocol |
After washing, the arrays were scanned with a GenePix4000B scanner (Molecular Devices, Sunnyvale, CA). The images were processed using GenePix Pro and the resulting text files were exported to Microsoft Excel.
|
| Description |
Comp 4_ST701-LMn(cy3) vs ST701-HMn(cy5)
Biological replicate 1 of 3
|
| Data processing |
The chips were normalized using the global normalization option within Genepix Pro. The data were filtered by removing all spots that were below the background noise or flagged as ‘bad’. Spots were considered to be below the background noise if the sum of the median intensities of the two channels was less than twice the highest average background of the chip.
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| |
|
| Submission date |
Jul 13, 2015 |
| Last update date |
Dec 08, 2015 |
| Contact name |
G Marcela Rodriguez |
| E-mail(s) |
[email protected]
|
| Phone |
9738543262
|
| Organization name |
Rutgers University
|
| Department |
Public Health Research Institute
|
| Street address |
225 Warren St
|
| City |
Newark |
| State/province |
NJ |
| ZIP/Postal code |
07103 |
| Country |
USA |
| |
|
| Platform ID |
GPL4057 |
| Series (1) |
| GSE70812 |
MntR(Rv2788): a transcriptional regulator that controls manganese homeostasis in Mycobacterium tuberculosis. |
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