|
| Status |
Public on Mar 08, 2015 |
| Title |
G. arboreum wild and wax mutant reg bio rep |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
G. arboreum wild plant
|
| Organism |
Gossypium arboreum |
| Characteristics |
organ: Leaf
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Jaakola et al. (2001), CTAB and PVP method was used for total RNA extraction.
|
| Label |
Cy3
|
| Label protocol |
For the aminoallyl labeling of cDNA, 45 µg of total RNA, DNA free, was taken and 2 µg of OligodT18 primer was added to it and final volume was made up to 18.0 µL with RNase-free water. The contents were mixed well and incubated at 70oC for 10 minutes. After incubation the mixture was placed on ice for 30 seconds, centrifuged and following contents were added: 5X First Strand buffer 6.0 µL 0.1 M DTT 3.0 µL 50X aminoallyl-dNTP mix 0.5 µL SuperScript III RT (200U/µL) 2.0 µL RNAse inhibitor 0.5 µL The contents were mixed well, centrifuged briefly and incubated at 42oC for 3 hours. Then to hydrolyze RNA, 2U of RNAse H were added, mixed and incubated at 37oC for 15 minutes. Added 25µL of 1M Tris (pH 6.8) to nutrilize pH. Unincorporated aa-dUTP and free amines were removed through (Qiagen pcr purification kit). Dry cDNA was dissolved in 5µl 0.1M sodium carbonate (pH 9.0) and 5 µl Cy3 dye was added to sample and mixed. The reaction was incubated for 1 hour in the dark at room temperature.
|
| |
|
| Channel 2 |
| Source name |
G. arboreum epicuticular wax mutant (Gawm3)
|
| Organism |
Gossypium arboreum |
| Characteristics |
organ: Leaf
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Jaakola et al. (2001), CTAB and PVP method was used for total RNA extraction.
|
| Label |
Cy5
|
| Label protocol |
For the aminoallyl labeling of cDNA, 45 µg of total RNA, DNA free, was taken and 2 µg of OligodT18 primer was added to it and final volume was made up to 18.0 µL with RNase-free water. The contents were mixed well and incubated at 70oC for 10 minutes. After incubation the mixture was placed on ice for 30 seconds, centrifuged and following contents were added: 5X First Strand buffer 6.0 µL 0.1 M DTT 3.0 µL 50X aminoallyl-dNTP mix 0.5 µL SuperScript III RT (200U/µL) 2.0 µL RNAse inhibitor 0.5 µL The contents were mixed well, centrifuged briefly and incubated at 42oC for 3 hours. Then to hydrolyze RNA, 2U of RNAse H were added, mixed and incubated at 37oC for 15 minutes. Added 25µL of 1M Tris (pH 6.8) to nutrilize pH. Unincorporated aa-dUTP and free amines were removed through (Qiagen pcr purification kit). Dry cDNA was dissolved in 5µl 0.1M sodium carbonate (pH 9.0) and 5 µl Cy3 dye was added to sample and mixed. The reaction was incubated for 1 hour in the dark at room temperature.
|
| |
|
| |
| Hybridization protocol |
Equal concentrations of Cy3 and Cy5 labelled cDNAs in terms of dye pmoles were taken and mixed in the 2X hybridization buffer. The target was heated to 95oC for three minutes and then immediately put on ice. Slide was placed in chamber of hybstation (Genomic Solutions®) with array side up. Target was injected to slide in chamber at 65oC and then slide was incubated at 42oC for 24 hours. Slide was removed from hybstation and washed in solution containing 1X SSC, 0.2% SDS at 42oC for 2 min with agitation. Then, slide was shifted to second wash solution containing 0.1X SSC, 0.2% SDS at room temperature and incubated for 2 min with agitation. After 2 min slide was moved to third wash solution containing 0.1X SSC and kept for 2 min with agitation. Slide was spin dried immediately by keeping slide in 50 ml tube with kimwipe stuffed in the bottom in a swing bucket rotor at 2000rpm for 1min and preceded for slide scan.
|
| Scan protocol |
Slide was scanned in Cy3 and Cy5 channels with the help of scanner UC 4X4 (Genomic solution®). The scanned slide was initially analyzed with the help of TIGER SPOTFINDER software available freely on line from the TM4 website.
|
| Description |
Un-sequenced PCR amplified DNAs from Clones were spotted in duplicate. The differentially expressed genes were after hybridization and data analysis sequenced.
|
| Data processing |
The data in the VALUE column were normalized with the help of house keeping genes and lowess normalization through MIDAS provided by TM4.
|
| |
|
| Submission date |
Mar 18, 2009 |
| Last update date |
Mar 08, 2015 |
| Contact name |
Muhammad Younas khan |
| E-mail(s) |
barozaikhan@gmail.com
|
| Phone |
+92-0333-7817319
|
| Organization name |
National centre of Excellence in Molecular biology
|
| Department |
Molecular biology
|
| Lab |
Plant genomics
|
| Street address |
Thokar Niaz Baig
|
| City |
Lahore |
| State/province |
Punjab |
| ZIP/Postal code |
54400 |
| Country |
Pakistan |
| |
|
| Platform ID |
GPL8266 |
| Series (1) |
| GSE15279 |
Studies of wax genes in cotton (Gossypium arboreum) |
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