|
| Status |
Public on Mar 01, 2010 |
| Title |
Oviparae-Females-rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Adult females
|
| Organism |
Acyrthosiphon pisum |
| Characteristics |
strain: LSR1
gender: female
reproduction mode: Oviparae
tissue: whole organism
|
| Treatment protocol |
Sexual individuals and sexuparae were produced by rearing the pea aphid at 18°C under a 12h photoperiod (short-day condition).
|
| Growth protocol |
The LSR1 clone of the pea aphid Acyrthosiphon pisum was reared and maintained as clonal individuals (parthenogenesis) on the plant Vicia fabae at 18°C under a 16h photoperiod for long-day condition (parthenogenesis). Sexual individuals and sexuparae were produced by rearing the pea aphid at 18°C under a 12h photoperiod (short-day condition).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were extracted from 10 adult females 48h after the final moult. Three independent samples were analysed for each reproductive morph. Total RNAs were extracted using miRNeasy purification kit (QIAGEN) according to manufacturer’s instructions. Quality of RNAs was checked with the Bioanalyser (Agilent). The assay started from 5 µg total RNA sample, which was size fractionated using a YM-100 Microcon centrifugal filter (Millipore) and the small RNAs (< 300 bases) isolated were 3’-extended with a poly(A) tail using poly(A) polymerase.
|
| Label |
Cy3
|
| Label protocol |
An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent dye staining; two different tags were used for the two RNA samples in dual-sample experiments (LC-Sciences).
|
| |
|
| Hybridization protocol |
Hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies). On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to target microRNA or other control RNAs and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 100 µL 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34 °C.
|
| Scan protocol |
Fluorescence images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics).
|
| Description |
n/a
|
| Data processing |
For each chip and each probe, the average signal value and its standard deviation were quantified. Data were analyzed by first subtracting the background, then integrating all the signals corresponding for the same probe for one given chip. A transcript to be listed as detectable must meets at least two conditions: signal intensity higher than 3x(background standard deviation) and spot CV < 0.5. CV. When repeating probes are present on an array, a transcript is listed as detectable only if the signals from at least 50% of the repeating probes are above detection level. Then normalization the signals from all arrays were performed using a LOWESS filter (Locally-weighted Regression). Results obtained in the different reproductive morphs were compared by comparing the ratio of the two sets of detected signals (log2) and p-values of the t-test were calculated. Differentially detected signals were those with p-value<0.05.
|
| |
|
| Submission date |
Jan 29, 2010 |
| Last update date |
Jan 29, 2010 |
| Contact name |
Stephanie Jaubert |
| E-mail(s) |
stephanie.jaubert@rennes.inra.fr
|
| Organization name |
INRA
|
| Lab |
UMR BiO3P
|
| Street address |
domaine de la Motte
|
| City |
Rennes |
| ZIP/Postal code |
35653 |
| Country |
France |
| |
|
| Platform ID |
GPL9993 |
| Series (2) |
| GSE20106 |
Phenotypic plasticity in the pea aphid Acyrthosiphon pisum: miRNA expression |
| GSE20110 |
Phenotypic plasticity in the pea aphid Acyrthosiphon pisum |
|
