|
| Status |
Public on Feb 06, 2016 |
| Title |
Heliothis virescens Gut-C4 |
| Sample type |
RNA |
| |
|
| Source name |
Heliothis virescens Gut-C4
|
| Organism |
Heliothis virescens |
| Characteristics |
tissue: gut
treatment: Control
|
| Growth protocol |
H. virescens larvae were provided by North Carolina State University and had been originally collected in Clayton, North Carolina, USA, in 1988 (JEN strain). H. virescens larvae were reared on pinto bean diet (Joyner and Gould, 1985) under laboratory conditions (26 °C, 55% relative humidity, 16:8 h light:dark photoperiod) in Jena, Germany. For feeding assays pinto bean diet was used. Newly molted 5th instar H. virescens larvae were reared for 3 days on artificial control diet and diet supplemented with gossypol, respectively, with following concentrations: 0.32% (w/v) and 0% (w/v) of gossypol. Larvae were kept at 27 °C during treatment. After weighing, larvae were anesthetized on ice and dissected into gut and rest of the body. Tissues were pooled according to their biological replicates (5 larvae and 5 guts, respectively, are one replicate, four biological replicates were prepared), snap frozen in liquid nitrogen and stored at -80 °C until RNA isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA isolation, tissues were ground to fine powder under liquid nitrogen. Total RNA was extracted by using the innuPREP RNA Mini Kit (Analytik Jena). RNA quality and integrity was assessed using RNA Nano chips on an Agilent 2100 Bioanalyzer (Agilent Technologies). The RNA quantity was determined using the NanoDrop ND-1000 spectrophotometer (Peqlab).
|
| Label |
Cy3
|
| Label protocol |
Agilent Technologies spike-in RNA was added to 100 ng of total RNA and labeled following the One-color Microarray-Based Gene Expression Analyisis (Low Input Quick Amp Labeling kit) protocol (Agilent Technologies). Labeled amplified cRNA samples were purified using Qiagen RNeasy Minikit 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 >1600 ng and the specific activity >6.0 pmol Cy3 per ug cRNA.
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| |
|
| Hybridization protocol |
The Cy3 labeled cRNA was hybridized to H. virescens (4x 180 K) microarray slides for 17 h at 65 °C in a hybridization oven. The microarray 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 manufacturers instruction, using an Agilent Microarray Scanner
|
| Description |
pooled from 5 individuals
|
| Data processing |
Data was extracted from TIFF images with Agilent Feature Extraction software version 9.1. Feature extracted data were analyzed using GeneSpring GX version 12.5 (Agilent Technologies) software. The data points were normalized between arrays to the median intensity and log base 2-transformation of the normalized data was performed.
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| |
|
| Submission date |
Feb 05, 2016 |
| Last update date |
Feb 06, 2016 |
| 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 |
GPL21432 |
| Series (1) |
| GSE77620 |
Heliothis virescens gene expression on gossypol suppelemented diet, larval gut and rest of body |
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