|
| Status |
Public on Jan 21, 2011 |
| Title |
Extraction method 2 pellet Poole_Ex2_Pellet |
| Sample type |
RNA |
| |
|
| Source name |
Extraction method 2 pellet Poole_Ex2_Pellet
|
| Organism |
Solanum lycopersicum |
| Characteristics |
strain: Ailsa Craig Solanum lycopersicum
tissue: Tomato pericarp
|
| Growth protocol |
Tomato plants were grown in M3 compost under glasshouse conditions at the University of Nottingham, Sutton Bonington Campus (UK, 52o47'N; 1o11'E). Mean day-time temperatures were maintained at 19°C, whilst night-time temperatures were maintained at 17°C. Side shoots were removed from the plants which were maintained in an upright form using crop supports.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Approximately 10-12 g was weighed and extracted with 2 ml/g i.e. 20-24 ml of Ex2 buffer ((0.2 M Tris-HCl pH 8.5, 0.2 M sucrose, 30 mM MgCL2, 0.6 M KCl) to which polyvinylpolypyrrolidone (1%) and beta-mercaptoethanol (0.31% v/v) had been added immediately before use) per g of tissue. Cell debris was removed by centrifugation at 10000 x g for 15 minutes. The pellet was retained for re-extraction using method Ex2. The supernatant was filtered through two layers of muslin, mixed with 0.1 vol 10% SDS and an equal volume of phenol: chloroform. The sample was vortexed vigorously for 45 seconds and centrifuged at 5000 x g for 5 minutes before the aqueous phase was transferred to a fresh tube. The aqueous phase was precipitated overnight at -20oC with an equal volume of isoproanol and 0.1 volumes of 3 M sodium acetate (pH 5.6). Precipitated nucleic acids were collected by centrifugation at 10000 X g for 10 minutes at 4oC. The supernatant was removed and the pellet was suspended again in 500µl of sterile distilled water, which was transferred to a sterile 1.5ml Eppendorf tube and precipitated material was removed by a brief, 3 second, pulse spin in a bench microcentrifuge. RNA was precipitated from the remaining solution by the addition of an equal volume of 8M lithium chloride followed by overnight storage at -20oC. RNA was recovered by centrifugation in a microcentrifuge at 16000 X g for 15 minutes and the supernatant was discarded. The RNA pellet was suspended again in 450µl of sterile distilled water and precipitated material was removed by centrifugation for 2 minutes. The supernatant was precipitated over night at -20oC with three volumes of ethanol and 0.1 volumes of 3 M sodium acetate (pH 5.6). RNA was recovered by centrifugation in a microcentrifuge at 16000 X g for 10 minutes. The pellet was washed with 1 ml of 80% ethanol, dried under vacuum and the RNA suspended in 60 µl of sterile distilled water.
|
| Label |
biotin
|
| Label protocol |
GeneChip arrays were stained with Streptavidin-Phycoerythrin solution as described by the Affymetrix GeneChip Expression Analysis Technical Manual (http://www.affymetrix.com).
|
| |
|
| Hybridization protocol |
Hybridisation was carried out as per the manufacturer's instructions as described in Affymetrix GeneChip Expression analysis technical manual (Affymetrix, www.affymetrix.com)
|
| Scan protocol |
GeneChip arrays were scanned with an Affymetrix G2500A GeneArray scanner, as described in the Affymetrix GeneChip Expression Analysis Technical Manual.
|
| Description |
'Cell debris' pellet extracted using the Ex2 extraction method
|
| Data processing |
Following scanning, non-scaled RNA signal intensity (CEL) files were generated using the GeneChip operating system (GCOS version 5.0; Affymetrix) as previously described. Robust Microarray Analysis (RMA) expression values were computed in Bioconductor using the language R.
|
| |
|
| Submission date |
Jul 21, 2009 |
| Last update date |
Jan 21, 2011 |
| Contact name |
Nottingham Arabidopsis Stock Centre (NASC) |
| E-mail(s) |
affy@arabidopsis.info
|
| Phone |
+44 (0)115 951 3237
|
| Fax |
+44 (0)115 951 3297
|
| URL |
http://arabidopsis.info/
|
| Organization name |
Nottingham Arabidopsis Stock Centre (NASC)
|
| Department |
School of Biosciences, University of Nottingham
|
| Street address |
Sutton Bonington Campus
|
| City |
Loughborough |
| ZIP/Postal code |
LE12 5RD |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL4741 |
| Series (1) |
| GSE17222 |
A tomato fruit RNA extraction method that isolates mRNAs encoding secreted and endomembrane associated proteins |
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