|
| Status |
Public on Jun 14, 2012 |
| Title |
Gut_CON/Ch3one_replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Gut, cholesterol diet (CON)
|
| Organism |
Helicoverpa zea |
| Characteristics |
developmental stage: 3rd instar larvae
tissue: midgut
treatment: cholesterol diet
|
| Treatment protocol |
For the microarray experiment, H. zea caterpillars were reared on the diets until they molted into the 3rd instar; shortly after molting, their midguts were removed and processed for RNA extraction. For midgut extraction, larvae were ice-cold anesthesized prior to dissection, which took place under phosphate-buffered saline.
|
| Growth protocol |
Helicoverpa zea eggs were acquired from Benzon Research, Inc. (Carlisle, PA), and the emerging hatchlings were used in the experiments. Insects were reared under laboratory conditions (26 °C, 55% RH, 16:8 hr = L:D) in College Station, Texas, USA on an artificial diet. The artificial diets used in the experiment is the diet described in Jing (2011), and is a hybrid of two diets: (1) the standard corn-soy-milk (CSM) diet used to rear many different caterpillar species (Cohen, 2004), and (2) a sterol-free diet developed by Ritter and Nes (Ritter and Nes, 1981a) for rearing H. zea. Some of the diet components in the CSM diet contain sterols, so these individual components were sterol-washed prior to use (Behmer and Grebenok, 1998). After the diets were mixed, they were suspended in a 1% agar mix (in a 1:6 dry to wet ratio) and presented to the developing caterpillars as food blocks.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Gut tissue was snap-frozen in liquid nitrogen. The frozen tissue was transferred to a liquid nitrogen-cooled mortar and ground. The homogenate was covered with 1 ml Trizol reagent (Invitrogen Corporation, Carlsbad, CA). Further steps were performed according to the manufacturer's instructions, replacing chloroform with 1-bromo-3-chloro-propane. Isolated RNA was quantified with a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE), while RNA quality was checked on a 1% agarose gel. Turbo DNase (Ambion) treatment was included to eliminate contaminating genomic DNA. The total RNA was further purified by using the RNeasy MinElute Clean up Kit (Qiagen) following the manufacturer's protocol. RNA integrity and quantity were verified on an Agilent 2100 Bioanalyzer, using RNA Nano chips (Agilent Technologies).
|
| Label |
Cy3
|
| Label protocol |
Agilent Technologies spike-in RNA was added to 500 ng of total RNA and labeled using a combination of the QuickAmp Labeling kit (Agilent Technologies) for two-color arrays following the manufacturer's instructions. Labeled amplified cRNA samples were purified using Qiagen RNeasy MinElute columns and analyzed on a Nanodrop ND-1000 spectrophotometer using the microarray function. Amplified cRNA samples were used for microarray hybridisation only if the yield was >825 ng and the specific activity >8.0 pmol Cy3 or Cy5 per ug cRNA.
|
| |
|
| Channel 2 |
| Source name |
Gut, cholestan-3-one diet (Ch3one)
|
| Organism |
Helicoverpa zea |
| Characteristics |
developmental stage: 3rd instar larvae
tissue: midgut
treatment: cholestan-3-one diet
|
| Treatment protocol |
For the microarray experiment, H. zea caterpillars were reared on the diets until they molted into the 3rd instar; shortly after molting, their midguts were removed and processed for RNA extraction. For midgut extraction, larvae were ice-cold anesthesized prior to dissection, which took place under phosphate-buffered saline.
|
| Growth protocol |
Helicoverpa zea eggs were acquired from Benzon Research, Inc. (Carlisle, PA), and the emerging hatchlings were used in the experiments. Insects were reared under laboratory conditions (26 °C, 55% RH, 16:8 hr = L:D) in College Station, Texas, USA on an artificial diet. The artificial diets used in the experiment is the diet described in Jing (2011), and is a hybrid of two diets: (1) the standard corn-soy-milk (CSM) diet used to rear many different caterpillar species (Cohen, 2004), and (2) a sterol-free diet developed by Ritter and Nes (Ritter and Nes, 1981a) for rearing H. zea. Some of the diet components in the CSM diet contain sterols, so these individual components were sterol-washed prior to use (Behmer and Grebenok, 1998). After the diets were mixed, they were suspended in a 1% agar mix (in a 1:6 dry to wet ratio) and presented to the developing caterpillars as food blocks.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Gut tissue was snap-frozen in liquid nitrogen. The frozen tissue was transferred to a liquid nitrogen-cooled mortar and ground. The homogenate was covered with 1 ml Trizol reagent (Invitrogen Corporation, Carlsbad, CA). Further steps were performed according to the manufacturer's instructions, replacing chloroform with 1-bromo-3-chloro-propane. Isolated RNA was quantified with a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE), while RNA quality was checked on a 1% agarose gel. Turbo DNase (Ambion) treatment was included to eliminate contaminating genomic DNA. The total RNA was further purified by using the RNeasy MinElute Clean up Kit (Qiagen) following the manufacturer's protocol. RNA integrity and quantity were verified on an Agilent 2100 Bioanalyzer, using RNA Nano chips (Agilent Technologies).
|
| Label |
Cy5
|
| Label protocol |
Agilent Technologies spike-in RNA was added to 500 ng of total RNA and labeled using a combination of the QuickAmp Labeling kit (Agilent Technologies) for two-color arrays following the manufacturer's instructions. Labeled amplified cRNA samples were purified using Qiagen RNeasy MinElute columns and analyzed on a Nanodrop ND-1000 spectrophotometer using the microarray function. Amplified cRNA samples were used for microarray hybridisation only if the yield was >825 ng and the specific activity >8.0 pmol Cy3 or Cy5 per ug cRNA.
|
| |
|
| |
| Hybridization protocol |
825 ng of each Cyanine 3- and Cyanine 5-labeled cRNA was used for each array. Hybridization was carried out at 65oC for 17 hours. Slides were washed in GE Wash Buffers according to the manufacturer's instructions (Agilent Technologies). Slides were treated in Stabilization and Drying Solution and scanned.
|
| Scan protocol |
As per manufacturer's instructions, using an Agilent microarray scanner.
|
| Description |
Pooled from 5 animals.
|
| Data processing |
Data was extracted from TIFF images with Agilent Feature Extraction software version 9.1. Raw data for both dye channels from output files were analyzed using GeneSpring microarray analysis software. The Sample data table includes the ratios (test/reference) log base 2 transformed normalized to median data. Data was averaged for structural and spike-in controls occurring several times in the microarray.
|
| |
|
| Submission date |
Jun 13, 2012 |
| Last update date |
Jun 14, 2012 |
| Contact name |
Heiko Vogel |
| E-mail(s) |
hvogel@ice.mpg.de
|
| Organization name |
Max Planck Institute for Chemical Ecology
|
| Department |
Dpt. of Entomology
|
| Street address |
Hans-Knoell-Strasse 8
|
| City |
Jena |
| ZIP/Postal code |
07745 |
| Country |
Germany |
| |
|
| Platform ID |
GPL14736 |
| Series (1) |
| GSE38699 |
Dietary sterols/steroids and the generalist caterpillar Helicoverpa zea: physiology, biochemistry and midgut gene expression |
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