|
| Status |
Public on Apr 07, 2016 |
| Title |
C3-C2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Control locust midgut/caeca sample replicate 3
|
| Organism |
Locusta migratoria |
| Characteristics |
tissue: midgut and caeca
diet: Control diet
|
| Treatment protocol |
After two days of normal feeding, individual 5th instar locusts received a single meal of 1.5% agar based artificial diet containing 2.4% Wesson salt mixture, 0.5% linoleïc acid, 0.6% cholesterol, 18.8% Vanderzant vitamin mixture, 50% cellulose, 14% dextrin, 8.1% caseïne, 2.8% peptone and 2.8% albumin. One group received a meal containing plant derived PI (SBTI, SBBI: 1% of total protein content), while another group received a control diet, where BSA was added to achieve equal total protein contents in the diets. 4 hours after ingestion of the meal, midgut and caeca samples were dissected for RNA extraction.
|
| Growth protocol |
Gregarious locusts were reared under crowded conditions with controlled temperature (32 ± 1°C), light (14h photoperiod) and relative humidity (40-60%). Locusts were fed daily with grass.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by using RNeasy Lipid Tissue Mini Kit (Qiagen) according to the manufacturer's instructions. In addition, a DNase treatment (RNase-free DNase set, Qiagen) was performed to eliminate potential genomic DNA contamination.
|
| Label |
Cy5
|
| Label protocol |
Labeling of the samples was performed by using the Quick Amp Labeling Kit (Two Color, Agilent Technologies), according to the manufacturer’s instructions. For each sample, 1µg total RNA was used for labeling. The fluorescently labeled samples were purified by means of the RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions.
|
| |
|
| Channel 2 |
| Source name |
Control locust midgut/caeca sample replicate 2
|
| Organism |
Locusta migratoria |
| Characteristics |
tissue: midgut and caeca
diet: Control diet
|
| Treatment protocol |
After two days of normal feeding, individual 5th instar locusts received a single meal of 1.5% agar based artificial diet containing 2.4% Wesson salt mixture, 0.5% linoleïc acid, 0.6% cholesterol, 18.8% Vanderzant vitamin mixture, 50% cellulose, 14% dextrin, 8.1% caseïne, 2.8% peptone and 2.8% albumin. One group received a meal containing plant derived PI (SBTI, SBBI: 1% of total protein content), while another group received a control diet, where BSA was added to achieve equal total protein contents in the diets. 4 hours after ingestion of the meal, midgut and caeca samples were dissected for RNA extraction.
|
| Growth protocol |
Gregarious locusts were reared under crowded conditions with controlled temperature (32 ± 1°C), light (14h photoperiod) and relative humidity (40-60%). Locusts were fed daily with grass.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by using RNeasy Lipid Tissue Mini Kit (Qiagen) according to the manufacturer's instructions. In addition, a DNase treatment (RNase-free DNase set, Qiagen) was performed to eliminate potential genomic DNA contamination.
|
| Label |
Cy3
|
| Label protocol |
Labeling of the samples was performed by using the Quick Amp Labeling Kit (Two Color, Agilent Technologies), according to the manufacturer’s instructions. For each sample, 1µg total RNA was used for labeling. The fluorescently labeled samples were purified by means of the RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions.
|
| |
|
| |
| Hybridization protocol |
The labeled samples were hybridized onto the microarrays according to the design described under 'Overall design', and by using the Gene Expression Hybridization Kit (Agilent Technologies) following the manufacturer's instructions. Hybridization was performed during 17 hr at 65°C. Next, the microarrays were washed twice in GE Wash Buffer 1 (Agilent) for 1 minute each, in GE Wash Buffer 2 (Agilent) at 37°C for 1 minute and in acetonitrile for 1 minute. Finally, the slides were immersed in Stabilization & Drying Solution (Agilent).
|
| Scan protocol |
Microarrays were scanned using a Genepix Personal 4100A confocal scanner (Axon Instruments) at a resolution of 5 µm and excitation wavelengths of 635 nm and 532 nm. The PMT voltages for each wavelength were adjusted to obtain an overall green/red ratio as close as possible to 1. Spot identification and quantification of the fluorescent signal was carried out using GenePix Pro 6.0 software (Axon Instruments).
|
| Description |
Control locust midgut/caeca sample replicate 3 vs Control locust midgut/caeca sample replicate 2
|
| Data processing |
The data obtained from all eight hybridizations were used for statistical analysis by means of the R package limma. Spots where the median foreground intensity was less than the average local background intensity (calculated over the complete microarray) plus twice its standard deviation on all arrays were deleted before analysis. Median intensities were background corrected using a normal-exponential convolution model (offset +50) and normalized by a Locally Weighed Scatterplot Smoothing (Lowess) function.
|
| |
|
| Submission date |
Apr 06, 2016 |
| Last update date |
Apr 07, 2016 |
| Contact name |
Jozef Vanden Broeck |
| E-mail(s) |
jozef.vandenbroeck@bio.kuleuven.be
|
| Organization name |
KU Leuven
|
| Department |
Animal Physiology and Neurobiology
|
| Lab |
Molecular Developmental Physiology and Signal Transduction
|
| Street address |
Naamsestraat 59
|
| City |
Leuven |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL11172 |
| Series (1) |
| GSE79995 |
Large scale transcript analysis of PI compensatory effects in the midgut and caeca of Locusta migratoria |
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