|
| Status |
Public on Apr 02, 2007 |
| Title |
Endosperm_LCM_4DAP_slideBG10_rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Siliques_4DAP_rep1
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
Developing siliques 4DAP. Prepared for LCM and mounted on glass slides. Tissue was scraped from the slide and used for RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was obtained from the tissue scrape samples using the Picopure RNA isolation kit (Arcturus) with optional on column DNase step (Qiagen). The purified total RNA was quantified using the Ribogreen RNA quantification kit (Invitrogen) according to the manufacturers’ instructions.
|
| Label |
Cy5
|
| Label protocol |
100ng of total RNA underwent a two round IVT amplification using the Message Amp II amino-allyl aRNA kit (Ambion) following the manufacturers’ instructions. Amino-allyl UTPs were incorporated into the second IVT reaction and coupled to Cy dyes (Amersham Biosciences) following the kits recommendations. All labelled aRNA targets were fragmented prior to hybridisation using Ambion fragmentation reagent
following the manufacturers’ protocol (Ambion).
|
| |
|
| Channel 2 |
| Source name |
Endosperm_4DAP_rep2
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
Developing siliques 4DAPwere prepared for LCM and mounted on glass slides. Endosperm was isolated from the sample using the Arcturus LM200 Pixcell II laser capture microdissection system.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was obtained from the endosperm sample using the Picopure RNA isolation kit (Arcturus) with optional on column DNase step (Qiagen). The purified total RNA was quantified using the Ribogreen RNA quantification kit (Invitrogen) according to the manufacturers’ instructions.
|
| Label |
Cy3
|
| Label protocol |
100ng of total RNA underwent a two round IVT amplification using the Message Amp II amino-allyl aRNA kit (Ambion) following the manufacturers’ instructions. Amino-allyl UTPs were incorporated into the second IVT reaction and coupled to Cy dyes (Amersham Biosciences) following the kits recommendations. All labelled aRNA targets were fragmented prior to hybridisation using Ambion fragmentation reagent
following the manufacturers’ protocol (Ambion).
|
| |
|
| |
| Hybridization protocol |
Pre-wash and BSA pre-hybridisation: Slides were pre-washed in 0.2% SDS for 2 mins and then washed twice in MilliQ (MQ) water. The slides were then placed in a 50mL Falcon tube containing fresh MQ water and incubated in a water bath for 20 mins at 50°C. Slides were then pre-hybridised in buffer containing 4x SSC, 0.5% SDS and 5% w/v bovine serum albumin (BSA, Sigma) at 42°C for 45 mins. Slides were then washed 5 times with MQ water at room temperature to remove BSA residue. Prior to hybridisation, the slides were dried by centrifuged at 5800 rpm.
Hybridisation: Hybridisations were carried out under LifterSlips (Erie scientific) placed over the region of the slide containing the array. A mix of SlideHyb buffer 1 (Ambion) and the labelled probes were then injected under the lifterslip. The arrays were then placed in a humidified hybridisation chamber (Corning) and hybridised over night in a 55°C water bath.
Post-hybridisation: The slides were removed from the hybridisation chamber and washed at 55°C for 8 mins with agitation in 50mL Falcon tubes containing 2x SSC, 0.1% SDS. The slides were then moved to new 50mL Falcon tubes and washed with constant agitation in 1x SSC, 0.1% SDS at room temperature for a further 6 mins. This procedure was repeated with a solution containing 0.5x SSC and then 0.1x SSC before the slides were dried by centrifugation at 5800 rpm.
|
| Scan protocol |
Scanning: Slide scanning was carried out using an Axon 4000B dual colour scanner running GenePix 4.1 software (Axon instruments).
|
| Description |
We hybridised whole silique total RNA against Endosperm total RNA obtained by laser capture microdissection.
|
| Data processing |
Genepix files were normalised by implementing elements of the SNOMAD gene analysis tools (http://pevsnerlab.kennedykrieger.org/snomadinput.html) at a local level using an splus script. This involved global mean normalisation, local mean
normalisation across the array surface and local mean normalisation across the element signal intensity.
|
| |
|
| Submission date |
Jan 05, 2007 |
| Last update date |
Jan 29, 2007 |
| Contact name |
Robert Day |
| E-mail(s) |
dayro345@student.otago.ac.nz
|
| Phone |
4797872
|
| Organization name |
University of Otago
|
| Department |
Biochemistry
|
| Lab |
Macknight
|
| Street address |
Cumberland Street
|
| City |
Dunedin |
| State/province |
Otago |
| ZIP/Postal code |
NA |
| Country |
New Zealand |
| |
|
| Platform ID |
GPL4720 |
| Series (2) |
| GSE6663 |
Expression analysis of proliferative phase Arabidopsis thaliana endosperm |
| GSE6703 |
Expression analysis of two-round IVT-amplified LCM samples: Arabidopsis endosperm |
|
