|
| Status |
Public on Sep 19, 2013 |
| Title |
Zea mays 2nd leaf control 60 min distal |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Zea mays 2nd leaf after 60min of volicitin treatment control
|
| Organism |
Zea mays |
| Characteristics |
line: B73
developmental stage: 3 week-old seedling
tissue: 2nd leaf
|
| Treatment protocol |
Zea mays (B73, 3 week old) were treated with volicitin (0.1nmol/microliter) in potassium phosphate buffer (pH 8, 50 mM) for 60 min. 1 nmol was applied per plant. Controls were untreated. Leaves segments (1cm) comprising the volicitin application site (local) and about 3cm leaf upwards (distal, also 1 cm) were cut and frozen in liquid N2 and stored at -80 °C prior to RNA extraction. Per leaf site (local and distal) 3 segments from 3 individual were pooled for one biological replicate. 2 biological replicates were performed per leaf site (local and distal, respectively).
|
| Growth protocol |
Zea mays (inbred line B73) plants were grown in soil (Redi Earth Plug and Seedling Mix, Sun Gro) in a growth chamber with a 12 h photoperiod, 60% relative humidity at 26°C for three weeks (V2 stage). Light intensity was set at app. 150 mmol m2 s-1.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The pooled leaf material (about 100mg) was used for RNA extraction. Total RNA was extracted with the Ultra Clean Plant RNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA) according to the manufacture’s instructions with the following modifications. Frozen plant samples were homogenized in 2 ml screw cap FastPrep tubes containing 0.5 g of Zirmil microbeads and 200 ml extraction buffer (PR1) for 20 sec at 6000 rpm in a Precellys tissue homogenizer (MO BIO Laboratories, Carlsbad, CA, USA). After this initial homogenization step the remaining 800 ml of PR1 were added and the sample again homogenized for 10 sec at 6000 rpm. The extract was then further processed as described in the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
1 μg RNA samples were used to make Cy3- and Cy5-labeled targets with a three step protocol as follows: 1) cDNA was synthesized using an oligo-(d)T primer that incorporated the T7 promoter; 2) targets were amplified using in vitro transcription to produce aminoallyl-labeled cRNA; and 3) aminoallyl-labeled cRNA was coupled to Cy-labeled dyes. Details of these protocols can be found at: http://www.maizearray.org/files/cRNA_Target_Production_Using_RNA_Amplification.pdf
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| |
|
| Channel 2 |
| Source name |
Zea mays_2nd leaf_60min_ volicitin_distal
|
| Organism |
Zea mays |
| Characteristics |
line: B73
developmental stage: 3 week-old seedling
tissue: 2nd leaf
|
| Treatment protocol |
Zea mays (B73, 3 week old) were treated with volicitin (0.1nmol/microliter) in potassium phosphate buffer (pH 8, 50 mM) for 60 min. 1 nmol was applied per plant. Controls were untreated. Leaves segments (1cm) comprising the volicitin application site (local) and about 3cm leaf upwards (distal, also 1 cm) were cut and frozen in liquid N2 and stored at -80 °C prior to RNA extraction. Per leaf site (local and distal) 3 segments from 3 individual were pooled for one biological replicate. 2 biological replicates were performed per leaf site (local and distal, respectively).
|
| Growth protocol |
Zea mays (inbred line B73) plants were grown in soil (Redi Earth Plug and Seedling Mix, Sun Gro) in a growth chamber with a 12 h photoperiod, 60% relative humidity at 26°C for three weeks (V2 stage). Light intensity was set at app. 150 mmol m2 s-1.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The pooled leaf material (about 100mg) was used for RNA extraction. Total RNA was extracted with the Ultra Clean Plant RNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA) according to the manufacture’s instructions with the following modifications. Frozen plant samples were homogenized in 2 ml screw cap FastPrep tubes containing 0.5 g of Zirmil microbeads and 200 ml extraction buffer (PR1) for 20 sec at 6000 rpm in a Precellys tissue homogenizer (MO BIO Laboratories, Carlsbad, CA, USA). After this initial homogenization step the remaining 800 ml of PR1 were added and the sample again homogenized for 10 sec at 6000 rpm. The extract was then further processed as described in the manufacturer’s instructions.
|
| Label |
Cy5
|
| Label protocol |
1 μg RNA samples were used to make Cy3- and Cy5-labeled targets with a three step protocol as follows: 1) cDNA was synthesized using an oligo-(d)T primer that incorporated the T7 promoter; 2) targets were amplified using in vitro transcription to produce aminoallyl-labeled cRNA; and 3) aminoallyl-labeled cRNA was coupled to Cy-labeled dyes. Details of these protocols can be found at: http://www.maizearray.org/files/cRNA_Target_Production_Using_RNA_Amplification.pdf
|
| |
|
| |
| Hybridization protocol |
Labelled cDNA hybridised using the MWG Gene-Frame system. Slides placed in shaking water bath for 16 h at 42 °C. Arrays then washed in 2x SSC (+0.1 % SDS), 1x SSC, 0.2x SSC and 0.1x SSC, each at 37 °C for 5 min prior to drying and scanning (http://ag.arizona.edu/microarray/Microarraymethod1.doc).
|
| Scan protocol |
Arrays were scanned using a GenePix Autoloader 4200AL.
|
| Data processing |
Raw data were extracted using Gene Pix Pro 6.0 program; then saved as gpr and txt files for each block individually (total 4 blocks on the chip resulting in 4 hybridizations). Within arrays, loess normalization was done using the Limma package in R (version R2.5.1) (Yang, Y. H., Dudoit, S., Luu, P., Lin, D. M., Peng, V., Ngai, J., and Speed, T. P. (2002). Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Research 30(4):e15). Between arrays, normalization was also done with the Limma package so that the scale the log-ratios to have the same median-absolute-deviation (MAD) across arrays (Smyth, G. K., and Speed, T. P. (2003). Normalization of cDNA microarray data. In: METHODS: Selecting Candidate Genes from DNA Array Screens: Application to Neuroscience, D. Carter (ed.). Methods Volume 31, Issue 4, December 2003, pages 265-273).
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| |
|
| Submission date |
Sep 18, 2013 |
| Last update date |
Sep 19, 2013 |
| Contact name |
Jurgen Engelberth |
| E-mail(s) |
jurgen.engelberth@utsa.edu
|
| Organization name |
University of Texas at San Antonio
|
| Department |
Biology
|
| Street address |
One UTSA Circle
|
| City |
San Antonio |
| State/province |
Texas |
| ZIP/Postal code |
78249 |
| Country |
USA |
| |
|
| Platform ID |
GPL6438 |
| Series (1) |
| GSE50981 |
Transcriptional changes in Zea mays seedlings treated with insect elicitor (volicitin). |
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