|
| Status |
Public on Nov 28, 2006 |
| Title |
Head Short Day Cy3 Rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Head of pea aphid reared under 12h photoperiod
|
| Organism |
Acyrthosiphon pisum |
| Characteristics |
Strain: YR2 (red holocyclic clone)
Parthenogenetic female
10 days of 12h photoperiod
Instar stage L3
Parthenogenetic female producing sexual individuals
|
| Biomaterial provider |
INRA Rennes, UMR 1099 BiO3P
|
| Treatment protocol |
Short Day reared pea aphids
|
| Growth protocol |
The clone YR2 of the pea aphid A. pisum was reared on broad bean (Vicia fabae) at 18°C. Parthenogenetic reproduction was maintained at 16 h of light and aphids were reared at low density (one to five individuals per plant) over three generations to prevent the production of winged forms. Conditions: 12 h of light (“short-day” or SD) that induced sexual forms after two generations. To initiate the experiment clonal third instar larvae (L3) were placed at SD photoperiod. This corresponds to generation G0. L3-G0 aphids reached adulthood (wingless adults or WA) and produced offspring (G1) that were transferred onto new plants and kept under SD conditions. A batch of 50 L3-G1 aphids was collected in the middle of the photophase (15:00) and immediately frozen in liquid nitrogen. As a control, few G1 aphids were maintained on plants to confirm the production of sexual forms in SD.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNAs were extracted from heads using either the RNeasy Mini kit (Qiagen, Germantown, MD, USA). RNAs were amplified by using MessageAmp aRNA kit (Ambion, Austin, TX, USA), starting with 2 µg of total RNA. RNAs were checked with a Bioanalyzer 2100 (Agilent Tech. Inc., Palo Alto, CA, USA), and quantified with a Nanodrop (Agilent).
|
| Label |
Cy3
|
| Label protocol |
5 µg of aRNA were labelled and purified using the CyScribe Post-labelling (anchored oligo-dT primed) and the CyScribe GFX Purification kits (Amersham Biosciences-GE, Fairfield, CT, USA)
|
| |
|
| Channel 2 |
| Source name |
Head of pea aphid reared under 16h photoperiod: reference channel
|
| Organism |
Acyrthosiphon pisum |
| Characteristics |
Strain: YR2 (red holocyclic clone)
Parthenogenetic female
10 days of 16h photoperiod
Instar stage L3
Female producing parthenogenetic females
|
| Biomaterial provider |
INRA Rennes, UMR 1099 BiO3P
|
| Treatment protocol |
Long Day reared pea aphids
|
| Growth protocol |
The clone YR2 of the pea aphid A. pisum was reared on broad bean (Vicia fabae) at 18°C. Parthenogenetic reproduction was maintained at 16 h of light and aphids were reared at low density (one to five individuals per plant) over three generations to prevent the production of winged forms. Conditions: 16 h of light (“long-day” or LD) that maintained parthenogenesis. To initiate the experiment clonal third instar larvae (L3) were placed under LD photoperiod. This corresponds to generation G0. L3-G0 aphids reached adulthood (wingless adults or WA) and produced offspring (G1) that were transferred onto new plants and kept under LD conditions. A batch of 50 L3-G1 aphids was collected in the middle of the photophase (15:00) and immediately frozen in liquid nitrogen. As a control, few G1 aphids were maintained on plants to confirm the production of asexual forms in LD.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNAs were extracted from heads using either the RNeasy Mini kit (Qiagen, Germantown, MD, USA). This Channel 2 sample corresponds to a mixture of total RNAs taken from the three replicated samples reared under Long Day photoperiod. RNAs were amplified by using MessageAmp aRNA kit (Ambion, Austin, TX, USA), starting with 2 µg of total RNA. RNAs were checked with a Bioanalyzer 2100 (Agilent Tech. Inc., Palo Alto, CA, USA), and quantified with a Nanodrop (Agilent).
|
| Label |
Cy5
|
| Label protocol |
5 µg of aRNA were labelled and purified using the CyScribe Post-labelling (anchored oligo-dT primed) and the CyScribe GFX Purification kits (Amersham Biosciences-GE, Fairfield, CT, USA).
|
| |
|
| |
| Hybridization protocol |
Hybridizations were performed in a Discovery XT System hybridization robot using the ChipMap 80 kit (Ventana Medical Systems, Tucson, AZ, USA) at the OUEST-Genopole and Agenae transcriptomic facilities (INRA Sribe, Rennes, France). Prehybridization was performed at 42ºC for 1 h in 2 ml of ChipSpread buffer containing 4xSSC and 0.2% SDS. Target cDNAs were mixed before hybridizations at 42ºC for 6 h (protocol N°2, ALC-D60/10-H48/8, Ventana). Washes were performed manually at room temperature in 1xSSC.
|
| Scan protocol |
Fluorescent images of the microarrays were generated with a Genepix 4000B scanner and Genepix Pro analysis software v5.0 (Axon Instruments, Molecular Devices Co., Sunnyvale, CA, USA).
|
| Description |
none
|
| Data processing |
Raw data were normalized by the MADSCAN software. After subtraction of the fluorescence background, a rank-invariant method and a spatial normalisation were performed before a scaling of the variance within each slide and between all the slides at the same rank. The normalized values were log-transformed and used to perform statistical analysis by GeneANOVA. The contribution of each experimental factor (Gene, Photoperiod, Biological replicate, Dye, Technical replicate) and the interaction between them was evaluated by a global ANOVA. Differentially expressed genes where selected by imposing stringent cut-offs on the local ANOVA graphic of the interaction “Gene-Photoperiod”
|
| |
|
| Submission date |
Nov 22, 2006 |
| Last update date |
Nov 27, 2006 |
| Contact name |
Denis TAGU |
| E-mail(s) |
denis.tagu@rennes.inra.fr
|
| Organization name |
INRA Rennes
|
| Department |
UMR 1099 BiO3P
|
| Street address |
BP35327
|
| City |
Le Rheu |
| ZIP/Postal code |
BP35653 |
| Country |
France |
| |
|
| Platform ID |
GPL4580 |
| Series (1) |
| GSE6363 |
Effect of day-length shortening on gene expression on pea aphid heads |
|
