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| Status |
Public on Apr 03, 2018 |
| Title |
C. elegans: total mRNA vs. AWA-specifc mRNA |
| Organism |
Caenorhabditis elegans |
| Experiment type |
Other
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| Summary |
Transcriptional profiling of AWA neurons comparing total mRNA of C. elegans
The aforementioned results provided us a clear indication that a GPCR expressed in AWA would likely be used for pheromone perception. To get a male AWA-specific expression profile, we adopted a poly-A-binding protein (PABP) mRNA pull-down approach to isolate cell-specific genes (Kunitomo et al., 2005) and co-transformed into the worms a sex determination gene, fem-3, to masculinize its nervous system. Ectopic expression of fem-3 in all neurons can change the entire nervous system to adopt a male identity (Ahringer & Kimble, 1991; Rosenquist & Kimble, 1988). We established the male AWA single-cell transcriptional profile by pooling mRNA from AWA with ectopic expression of PABP there driven by an AWA-specific promoter from odr-7. This PABP fused with an epitope protein tag (FLAG tag) is then extracted using the corresponding antibody, while AWA enriched transcripts with poly-A tail, including the GPCRs, were pulled down together and subjected to microarray analysis. The results confirmed the existence of dyp-13 (hypodermal gene), myo-3 (muscle gene) and odr-10 (AWA-specific over-expression gene) in total RNA samples while odr-10 was found to be over-expressed in AWA enriched sample, where dpy-13 and myo-3 were depleted. The pull-down RNA sample was labeled with cyanine 5 (which is excited by a 532nm laser) and the total RNA sample was labeled with cyanine 3 (which is excited by a 633nm laser). Comparison between transcriptome profiles revealed key differences between whole worm and male AWA neurons, which allows identification of male AWA-enriched transcripts including those encoding GPCRs.
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| Overall design |
Two-condition experiment, AWA-specific pull-down mRNA vs. total mRNA. Biological replicates: 4 control replicates, 4 AWA-specific pull-down replicates.
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| Contributor(s) |
Chow KL, Zhou Y |
| Citation missing |
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| Submission date |
Apr 02, 2018 |
| Last update date |
Apr 04, 2018 |
| Contact name |
king L. Chow |
| E-mail(s) |
bokchow@ust.hk
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| Phone |
23587342
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| Organization name |
HKUST
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| Department |
Life Science
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| Lab |
King L. CHOW
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| Street address |
clear water bay
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| City |
Hong Kong |
| State/province |
Hong Kong |
| ZIP/Postal code |
000000 |
| Country |
Hong Kong |
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| Platforms (1) |
| GPL13922 |
Axon/Agilent-015061:C. elegans Oligo Microarray 4x44K |
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| Samples (4)
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| GSM3074024 |
AWA-specific pull-down mRNA Replicate 1 |
| GSM3074025 |
AWA-specific pull-down mRNA Replicate 2 |
| GSM3074026 |
AWA-specific pull-down mRNA Replicate 3 |
| GSM3074027 |
AWA-specific pull-down mRNA Replicate 4 |
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| Relations |
| BioProject |
PRJNA448452 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE112610_RAW.tar |
60.6 Mb |
(http)(custom) |
TAR (of TXT) |
| Processed data included within Sample table |
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