|
| Status |
Public on Apr 28, 2015 |
| Title |
Dye Flip of DTT_3hrs/Control, biological rep1 of 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
M.tb in Log phase, DTT
|
| Organism |
Mycobacterium tuberculosis H37Rv |
| Characteristics |
treatment: DTT_3hrs
|
| Treatment protocol |
To the test sample added 1 mM DTT (reduced) for 3hrs.
|
| Growth protocol |
M.tb H37 Rv culture was grown upto log pahse.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol method was used for RNA isolation as per manufactures protocol.
|
| Label |
cy3
|
| Label protocol |
I. 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 a microcon10 column (YM10 from Millipore) with 400µl TE and spin for 10 min at 10,000 Xg. Empty flow-through in collection tube and return primed column. Add both reactions (cy3 and cy5) to column and centrifuge at 10,000 Xg until ~25µl remaining (~20-30 min). Wash with 200µl TE and spin until almost dry (<5µl). 6. Recover cDNA by inverting microcon into new collection 1.5 ml tube and centrifuge at half max speed 1 min. 7. Bring sample to 5µl with TE. The detailed labeling and hybridization protocol can be obtained at http://www.cag.icph.org/downloads_page.htm#LabelingProtocols.
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| |
|
| Channel 2 |
| Source name |
M.tb in Log phase, untreated
|
| Organism |
Mycobacterium tuberculosis H37Rv |
| Characteristics |
treatment: Untreated Control
|
| Treatment protocol |
To the test sample added 1 mM DTT (reduced) for 3hrs.
|
| Growth protocol |
M.tb H37 Rv culture was grown upto log pahse.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol method was used for RNA isolation as per manufactures protocol.
|
| Label |
cy5
|
| Label protocol |
I. 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 a microcon10 column (YM10 from Millipore) with 400µl TE and spin for 10 min at 10,000 Xg. Empty flow-through in collection tube and return primed column. Add both reactions (cy3 and cy5) to column and centrifuge at 10,000 Xg until ~25µl remaining (~20-30 min). Wash with 200µl TE and spin until almost dry (<5µl). 6. Recover cDNA by inverting microcon into new collection 1.5 ml tube and centrifuge at half max speed 1 min. 7. Bring sample to 5µl with TE. The detailed labeling and hybridization protocol can be obtained at http://www.cag.icph.org/downloads_page.htm#LabelingProtocols.
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| |
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| |
| Hybridization protocol |
II. 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 GenePix4200AL scanner (Molecular Devices, Sunnyvale, CA). The images were processed using GenePix Pro and the resulting text files were exported to Microsoft Excel.
|
| Description |
Dye Flip of 1 of 3
DTT_3 hrs_1_1
|
| Data processing |
The chips were normalized by the print-tip Lowess method (Dudoit, 2000). 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. The ratio of the average median intensity of Cy5 over the average median intensity of Cy3 was determined for each spot. The data was then processed for statistical significance using Partek Genomics Suite ver 6.5. A false discovery rate (FDR) of 5% was set as cut-off to select for significantly differentially expressed genes from the normalized and processed expression data
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| |
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| Submission date |
Apr 28, 2015 |
| Last update date |
Apr 28, 2015 |
| Contact name |
Ashwani Kumar |
| E-mail(s) |
ashwanik@imtech.res.in
|
| Phone |
919216530070
|
| Organization name |
CSIR-Institute of Microbial Technology
|
| Department |
Molecular Microbiology
|
| Street address |
Sec 39A
|
| City |
Chandigarh |
| State/province |
Chandigarh |
| ZIP/Postal code |
160036 |
| Country |
India |
| |
|
| Platform ID |
GPL4057 |
| Series (1) |
| GSE68350 |
Expression analysis of M.tb H37Rv after treatment with DTT (reduced) for 3 hrs |
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