|
| Status |
Public on Aug 17, 2013 |
| Title |
37I-rep2 vs 74NI-rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
L4637 inoculated kernels
|
| Organism |
Zea mays |
| Characteristics |
inbred line: L4637
inoculation status: inoculated
tissue: kernels and silks
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Kernels of each inoculated and non-inoculated inbred were milled in an IKA A11 basic Analytical mill. Total RNA was extracted from 0.5 g of mill kernels and silks using the Trizol protocol following 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 |
L4674 non inoculated kernels
|
| Organism |
Zea mays |
| Characteristics |
inbred line: L4674
inoculation status: non inoculated
tissue: kernels and silks
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Kernels of each inoculated and non-inoculated inbred were milled in an IKA A11 basic Analytical mill. Total RNA was extracted from 0.5 g of mill kernels and silks using the Trizol protocol following 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 |
Hybridization mix 1. Mix the following in a microfuge tube: 20X SSC 6.0uL Liquid Block 3.6uL 2% SDS 2.4uL Both Labeled Targets --- uL H2O to 60 uL Note:- We recommend using 48 pmol of Cy5 labeled targets and 48 pmol of Cy3 labeled targets in a 60 μL hybridization volume, but this amount should be optimized empirically. We have used up to 100 pmol target with Arabidopsis. However, it is important not to add too high an amount of labeled targets in to the hybridization mix, since eventually this results in Cy-dye precipitation, leading to a very high background. 2. Denature labeled target by incubating tube at 65o C for 5 min. 3. Transfer tube to ice immediately or apply on to the slides directly. 4. Rinse ArrayIt™ Hybridization Cassette with distilled water and dry thoroughly. 5. Make sure flexible rubber gasket is seated evenly in gasket channel. 6. Add 15 μL water to the lower groove inside the cassette chamber. 7. Insert the microarray (1 x 3 or 25mm x 75mm slide) into cassette chamber, DNA side up. 8. Place the lifter slip over the microarray slide (make sure the white stripe of the lifterslip is at the lower side) 9. Apply the PRE-HEATED sample slowly to the one end of the lifterslip and let it disperse. 10. Quickly place the clear plastic cassette lid on top of the cassette chamber. 11. Apply downward pressure and manually tighten (clockwise) the four sealing screws. 12. Check all four screws again to confirm a tight seal. 13. Place the cassette into a hybridization oven set at 55oC. 14. Allow the hybridization reaction to proceed for 8-12 h. 15. After hybridization, remove cassette, manually loosen the four sealing screws (counterclockwise) and remove lid. 16. Remove the microarray slide from the cassette chamber using forceps and place the slides into the washing buffer. Note:- Our recipe (SSC buffer, SDS, and Liquid Block) provides a low background, high sensitivity, and highly-reproducible results. Commercially-available hybridization buffers containing 50% formamide can also be used, in which case hybridization should be done at 42 oC instead of 55 oC.
|
| Scan protocol |
Protocols used for cDNA labeling, hybridization and microarray scanning can be obtained in http://www.maizearray.org/maize_protocols.shtml
|
| Description |
Sample name: 146
|
| Data processing |
The median foreground values for each channel were first normalized using the lowess method of the limma package in R (within each array) and then using limma’s quantile method (between all arrays) (Smyth GK: Limma: linear models for microarray data. In Bioinformatics and Computational Biology Solutions using R and Bioconductor. Edited by: Gentleman R, Carey V, Dudoit S, Irizarry R, Huber W. New York: Springer; 2005:397-4).
Probes with expression values more than 3.0 standard deviations above the average foreground intensity of the negative controls in the arrays were included for the analysis. For differential expression, we used the unadjusted p-value generated by limma. Differentially expressed probes were identified using 1.2 fold cutoff for expression ratios with a limma assigned p-value < 0.05. Normalized data were log2 transformed and then fitted into mixed model ANOVAs (normalized.txt, linked as supplementary file on Series record). Estimates of the expression differences were calculated using the mixed model. Based on these statistical analyses, the spots with tests with an FDR less than or equal to 5% and with changes in signal intensity between each comparison of 1.2-fold or higher were considered as differentially expressed.
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| |
|
| Submission date |
Aug 22, 2012 |
| Last update date |
Aug 17, 2013 |
| Contact name |
Valeria Alina Campos Bermudez |
| E-mail(s) |
[email protected], [email protected]
|
| Organization name |
CEFOBI
|
| Street address |
Suipacha 531
|
| City |
Rosario |
| ZIP/Postal code |
2000 |
| Country |
Argentina |
| |
|
| Platform ID |
GPL6438 |
| Series (1) |
| GSE40288 |
Maize kernels: Non Inoculated vs. Inoculated with Fusarium verticillioides |
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