|
| Status |
Public on Sep 15, 2010 |
| Title |
Nigriventris_small_male_repl1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Nigriventris small male abdominal epidermis
|
| Organism |
Onthophagus nigriventris |
| Characteristics |
species: Onthophagus nigriventris
sex: male
morph: small, sneaker
developmental stage: pupae within 24hrs after pupation
tissue: dissected dorsal abdominal epidermis
sample type: tissue pooled from 4 individuals
|
| Treatment protocol |
Wild type (unmanipulated) animals were used.
|
| Growth protocol |
All animals were reared under controlled laboratory conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from dissected tissue using Qiagen RNeasy kit according to the manufacturers protocol.
|
| Label |
Cy5
|
| Label protocol |
RNA was amplified using a T7-based RNA amplification method (Klebes et al. 2002 (PMID 12186645)) as follows. One microgram of RNA was reverse transcribed using Oligo(dT)-T7 primer (Ambion) and SuperScriptIII reverse transcriptase (Invitrogen). Second strand synthesis was performed using DNA polymerase (Invitrogen) and RNase H (Invitrogen). In-vitro transcription was performed using the MEGAscript T7 kit (Ambion). Amplified RNA (aRNA) was labeled with Cy5 using the ULS aRNA Fluorescent Labeling Kit (Kreatech, Amsterdam, Netherlands).
|
| |
|
| Channel 2 |
| Source name |
Nigriventris small male thoracic epidermis
|
| Organism |
Onthophagus nigriventris |
| Characteristics |
species: Onthophagus nigriventris
sex: male
morph: small, sneaker
developmental stage: pupae within 24hrs after pupation
tissue: dissected dorsal prothoracic epidermis
sample type: tissue pooled from 4 individuals
|
| Treatment protocol |
Wild type (unmanipulated) animals were used.
|
| Growth protocol |
All animals were reared under controlled laboratory conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from dissected tissue using Qiagen RNeasy kit according to the manufacturers protocol.
|
| Label |
Cy3
|
| Label protocol |
RNA was amplified using a T7-based RNA amplification method (Klebes et al. 2002 (PMID 12186645)) as follows. One microgram of RNA was reverse transcribed using Oligo(dT)-T7 primer (Ambion) and SuperScriptIII reverse transcriptase (Invitrogen). Second strand synthesis was performed using DNA polymerase (Invitrogen) and RNase H (Invitrogen). In-vitro transcription was performed using the MEGAscript T7 kit (Ambion). Amplified RNA (aRNA) was labeled with Cy3 using the ULS aRNA Fluorescent Labeling Kit (Kreatech, Amsterdam, Netherlands).
|
| |
|
| |
| Hybridization protocol |
Labeled aRNAs from test and reference samples (adjusted to a total of 60pmol dye for each) were mixed with KREAblock (ULS aRNA Fluorescent Labeling kit, Kreatech, Amsterdam, Netherlands) and 2x enhanced cDNA hybridization buffer (Genisphere), and then heated at 80°C for 10 min. Microarrays were pre-treated for one hour at 55°C in pre-hybridization buffer consisting of 5xSSC, 0.1%SDS, and 1% I-block (Applied Biosystems, CA). Both mixed sample and the microarrays were kept at 55°C until hybridization. Hybridization was performed in a dark humidified chamber at 55°C overnight. Microarrays were rinsed in buffer A (2xSSC/0.2%SDS) at 55°C and then incubated in buffer A at 65°C for 10min. Arrays were then transferred to 2xSSC (room temperature) for 10 min, followed by incubation in 0.2x SSC for 10min (room temperature). Arrays were then dried by centrifugation at 500rcf for 4 min.
|
| Scan protocol |
Microarrays were scanned using a GenePix scanner 4200 (Molecular Devices).
|
| Description |
This project sought to identify shared and divergent patterns of gene expression between two male morphs (horned/fighter and hornless/sneaker) of horned beetles in the genus Onthophagus. We were interested in whether morph-biased expression was as divergent as sex-biased expression, so we also surveyed patterns of gene expression in females. We investigated gene expression in three epidermal tissues (head horn, thoracic horn and legs) with respect to dorsal abdomindal epidermis, which does not express outgrowths. We also surveyed gene expression in the central brain, relative to neural ganglia.
|
| Data processing |
Data were filtered and extracted using GenePix v.5.0 and imported into Bioconductor. Normalization for M-values was performed using spatial and intensity dependent Lowess smoothing in OLIN and differential expression was assessed using Limma.
|
| |
|
| Submission date |
Aug 04, 2010 |
| Last update date |
Sep 03, 2010 |
| Contact name |
Emilie Snell-Rood |
| E-mail(s) |
emcsnell@indiana.edu
|
| URL |
http://www.indiana.edu/~ecsnellr/
|
| Organization name |
Indiana University
|
| Department |
Biology
|
| Lab |
Moczek
|
| Street address |
915 E 3rd St MY150
|
| City |
Bloomington |
| State/province |
IN |
| ZIP/Postal code |
47405 |
| Country |
USA |
| |
|
| Platform ID |
GPL7555 |
| Series (1) |
| GSE23425 |
Developmental decoupling of alternative phenotypes: insights from the transcriptomes of horn-polyphenic beetles |
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