|
| Status |
Public on Jun 12, 2004 |
| Title |
EV24, Wild-type versus ddm1/+ backcross mRNA |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
ddm1/+ poly(A)+ mRNA
|
| Organism |
Arabidopsis thaliana |
| Extracted molecule |
total RNA |
| |
|
| Channel 2 |
| Source name |
Wild-type poly(A)+ mRNA
|
| Organism |
Arabidopsis thaliana |
| Extracted molecule |
total RNA |
| |
|
| |
| Description |
Wild-type and ddm1/+ backcrossed bolting plants
Total RNA was extracted with Trizol (Invitrogen) from plants, following the manufacturer's instructions. Polyadenylated RNA were then extracted from total RNA with either the MicroPoly(A)purist (Ambion) or the Dynabeads kit (oligo(dT)25, Dynal). For each hybridization experiment, 1ug of poly(A)+ WT and ddm1 RNA were labeled using an indirect labeling procedure. The probes were synthesized from poly(A)+ RNA using random and oligodT primers and Superscript II reverse transcriptase (Invitrogen) to incorporate amine-modified nucleotides (amino allyl-dUTP; Sigma). cDNA samples were purified, concentrated using Microcon 30 spin columns (Millipore) and dried.
Dried cDNA were resuspended in Sodium Bicarbonate buffer 0.05M, pH 9.0 and coupled with Cy3 (Wild-type) or Cy5 (ddm1/+) amine reactive dyes (Amersham). The labeled cDNA were purified using the Qiagen Qiaquick PCR purification kit and concentrated with Microcon 30 spin columns.
For hybridization, the sample volume was brought to 25ul with a final concentration of 4X SSC, 0.2% SDS. Microarrays were hybridized at 65°C, for approximately 15 hr and washed once in 1XSSC, 0.2%SDS and twice in 0.2XSSC.
Slides were pre-hybridized in BSA 1%, SDS 0.1% for 1 hour before hybridization.
Slides were then scanned with a GenePix 4000A scanner (Axon Instruments). We quantified the fluorescence with the GenePix Pro software.
Sample_series_id: EV23+24
|
| |
|
| Submission date |
Apr 21, 2004 |
| Last update date |
Oct 28, 2005 |
| Contact name |
Matthew Wayne Vaughn |
| E-mail(s) |
vaughn@tacc.utexas.edu
|
| Phone |
(512) 232-7124
|
| Organization name |
University of Texas at Austin
|
| Department |
Texas Advanced Computing Center
|
| Lab |
Vaughn
|
| Street address |
10100 Burnet Rd
|
| City |
Austin |
| State/province |
TX |
| ZIP/Postal code |
78758 |
| Country |
USA |
| |
|
| Platform ID |
GPL785 |
| Series (2) |
| GSE1324 |
EV23+24 mRNA levels in Wild-type versus ddm1/+ backcross bolting Arabidopsis thaliana plants |
| GSE1354 |
mRNA levels in Wild-type versus ddm1 Arabidopsis thaliana |
|
| Data table header descriptions |
| ID_REF |
Spot name |
| F635_MEDIAN |
Cy5 channel foreground median |
| F635_MEAN |
Cy5 channel foreground mean |
| F635_SD |
Cy5 channel foreground stdev |
| B635_MEDIAN |
Cy5 channel background median |
| B635_MEAN |
Cy5 channel background mean |
| B635_SD |
Cy5 channel background stdev |
| PCT_GT_B635+1SD |
Percent Cy5 channel background > 1 stdev |
| PCT_GT_B635+2SD |
Percent Cy5 channel background > 2 stdev |
| F532_MEDIAN |
Cy3 channel foreground median |
| F532_MEAN |
Cy3 channel foreground mean |
| F532_SD |
Cy3 channel foreground stdev |
| B532_MED |
Cy3 channel background median |
| B532_MEAN |
Cy3 channel background mean |
| B532_SD |
Cy3 channel background stdev |
| PCT_GT_B532+1SD |
Percent Cy3 channel background > 1 stdev |
| PCT_GT_B532+2SD |
Percent Cy3 channel background > 2 stdev |
| VALUE |
Natural log of ratio of means |
| F635_MEDIAN-B635 |
Foreground median Cy5 - Background Cy5 |
| F532_MEDIAN-B532 |
Foreground median Cy3 - Background Cy3 |
| F635_MEAN-B635 |
Foreground mean Cy5 - Background Cy5 |
| F532_MEAN-B532 |
Foreground mean Cy3 - Background Cy3 |
| FLAGS |
Present:0, Absent:-50 |
