|
| Status |
Public on Jul 24, 2007 |
| Title |
Medicago truncatula 3 dpi with Sinorhizobium meliloti strain 1021 exoY mutant vs strain 1021 wild type. rep5 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Medicago truncatula 3 days post-inoculation with Sinorhizobium meliloti strain 1021 wild type. rep5
|
| Organism |
Medicago truncatula |
| Characteristics |
Medicago truncatula; lines - A17; tissue- roots; age - 8 days; treatment - 3 days after Sinorhizobium meliloti strain 1021 wild type inoculation
|
| Growth protocol |
Medicago truncatula seeds from each line were germinated as previously described (Penmetsa and Cook, 2000). M. truncatula cv. Jemalong A17 seeds were isolated from crushed pods and scarified in concentrated sulfuric acid for 10-12 minutes {Penmetsa, 2000 #5901}. Scarified seeds were rinsed > 6 times in sterile distilled water, incubated at 4oC overnight, rinsed > 6 times in sterile distilled water and germinated 1 day on 1% agar plates (Sigma, A7921), turned vertically and placed in the dark. Germinated seedlings (30-50 per plate) were placed on 25 cm x 25 cm Buffered Nod Media (BNM) pH 6.5, 1.5% agar plates. {Engstrom, 2002 #2626}. BNM plates were supplemented with 1mM of the ethylene synthesis inhibitor a-aminoisobutyric acid (AIB) to facilitate nodulation {Satoh, 1980 #5903; Peters, 1989 #5909; Engstrom, 2002 #2626}. Seedlings were grown at 25oC under fluorescent lights in a 16:8 hour light:dark cycle for 5 days with roots covered by foil. One day before inoculation, any roots that had grown down into the agar were placed back on top of the agar. Roots were inoculated 5 days after transfer to BNM plates. Saturated bacterial cultures were washed 2-3 times in 1/2x BNM pH 6.5, and resuspended at OD600 = 0.05. Twenty-five mLs of the appropriate inoculum was applied to each 25 x 25 cm plate. After harvesting of roots, at least 5 plants were left on each plate and observed for 1 month to verify nodulation phenotype and lack of contamination. Roots were harvested 3 days after inoculation with bacterial suspensions. Roots from >50 plants per experiment were dipped in liquid nitrogen and then cut away from the shoot and from the bottom 0.5 cm of the root tip. Roots were frozen at -80oC until RNA was prepared
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using the RNAeasy Plant Mini Kit (Qiagen 74904). RNA was DNased with RNase-free DNase (Qiagen 79254). DNased RNA was pooled from 5 separate experiments for each S. meliloti inoculum strain treatment.
|
| Label |
Cy5
|
| Label protocol |
A 66 mg aliquot of each pooled RNA sample was used for cDNA synthesis with random primers using a 3DNA Array 50 Expression Array Detection Kit (Genisphere). One half of the aliquot was labeled during cDNA synthesis with Cy3 dye while the other half was labeled with Cy5 dye using a 3DNA Array 900 Expression Array Detection Kit for cDNA Microarrays (Genisphere, Inc.,Hatfield, PA). cDNA was synthesized following instructions from 3DNA Array 900 Expression Array Detection Kit Appendix A (Genisphere Inc.).
|
| |
|
| Channel 2 |
| Source name |
Medicago truncatula 3 days post-inoculation with Sinorhizobium meliloti strain 1021 exoY mutant. rep5
|
| Organism |
Medicago truncatula |
| Characteristics |
Medicago truncatula; lines - A17; tissue- roots; age - 8 days; treatment - 3 days after Sinorhizobium meliloti strain 1021 exoY inoculation
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using the RNAeasy Plant Mini Kit (Qiagen 74904). RNA was DNased with RNase-free DNase (Qiagen 79254). DNased RNA was pooled from 5 separate experiments for each S. meliloti inoculum strain treatment.
|
| Label |
Cy3
|
| Label protocol |
A 66 mg aliquot of each pooled RNA sample was used for cDNA synthesis with random primers using a 3DNA Array 50 Expression Array Detection Kit (Genisphere). One half of the aliquot was labeled during cDNA synthesis with Cy3 dye while the other half was labeled with Cy5 dye using a 3DNA Array 900 Expression Array Detection Kit for cDNA Microarrays (Genisphere, Inc.,Hatfield, PA). cDNA was synthesized following instructions from 3DNA Array 900 Expression Array Detection Kit Appendix A (Genisphere Inc.).
|
| |
|
| |
| Hybridization protocol |
Microarrays for each time point were hybridized to cDNAs from both Al- treated and control roots, with cDNAs from the two different treatments labeled with Cy5 and Cy3 dyes. Each hybridization was repeated at least six times to account for technical variability, with triplicates of each dye combination to control for dye effects. A modified two-step hybridization reaction was performed as described in the 3DNA Array 900 Expression Array Detection Kit (Genisphere).
|
| Scan protocol |
Microarray slides were scanned using an GenePix4000B (Axon) two-laser scanner and image analysis was performed using GenePix 5.0 (Axon) software
|
| Description |
comparison of M.truncatula roots at 3 days post-inoculation with Sinorhizobium meliloti strain 1021 exoY mutant with roots at 3 days post-inoculation with Sinorhizobium meliloti strain 1021 wild type . rep5
|
| Data processing |
Background-subtracted mean intensities for both tissues were log2 transformed and normalized using lowess and ANOVA
|
| |
|
| Submission date |
Jul 18, 2007 |
| Last update date |
Jul 18, 2007 |
| Contact name |
Kathryn A VandenBosch |
| E-mail(s) |
vande102@umn.edu
|
| Phone |
+1 612-624-2755
|
| Organization name |
University of Minnesota
|
| Department |
Plant Biology
|
| Lab |
Kate VandenBosch
|
| Street address |
Room 250 BioSci 6022 1445 Gortner Ave
|
| City |
St Paul |
| State/province |
MN |
| ZIP/Postal code |
55108 |
| Country |
USA |
| |
|
| Platform ID |
GPL4799 |
| Series (1) |
| GSE8509 |
Responses in Medicago truncatula to Sinorhizobium meliloti wild type or the succinoglycan-deficient exoY mutant. |
|
| Data table header descriptions |
| ID_REF |
|
| VALUE |
-[INV_VALUE] |
| F635 Mean |
Channel 1 mean intensity |
| F635 Median |
Channel 1 median intensity |
| F635 SD |
Channel 1 mean standard deviation |
| B635 Mean |
Channel 1 mean background intensity |
| B635 Median |
Channel 1 median background intensity |
| B635 SD |
Channel 1 background standard deviation |
| % > B635+2SD |
Percent of feature pixels that were greater than two standard deviations of |
| F532 Mean |
Channel 2 mean intensity |
| F532 Median |
Channel 2 median intensity |
| F532 SD |
Channel 2 mean standard deviation |
| B532 Mean |
Channel 2 mean background intensity |
| B532 Median |
Channel 2 median background intensity |
| B532 SD |
Channel 2 background standard deviation |
| % > B532+2SD |
Percent of feature pixels that were greater than two standard deviations of the background over the background signal |
| Flags |
0 denotes satisfactory features, while <0 denotes features that did not meet |
| INV_VALUE |
Lowess normalized log2 ratio of means defined as CH1 divided by CH2 |
