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| Status |
Public on May 14, 2008 |
| Title |
Two distinct mechanisms generate endogenous siRNAs from bidirectional transcription in Drosophila melanogaster. |
| Organism |
Drosophila melanogaster |
| Experiment type |
Expression profiling by array
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| Summary |
Cis-natural antisense transcripts (cis-NATs) have been speculated to be substrates for endogenous RNA interference (RNAi), but little experimental evidence for such a pathway in animals has been reported. Analysis of massive Drosophila melanogaster small RNA data sets now reveals that endogenous small interfering RNAs (siRNAs) are produced via bidirectional transcription. >100 cis-NATs with overlapping 3' exons generate 21-nt, Dicer-2 (Dcr-2)dependent, 3'-end modified siRNAs.
To determine whether any co-expressed cisNATs are denied entry into the RNAi pathway, we analyzed the gene expression profile of S2 cells. The analysis suggested that the processing of cis-NATs by RNAi are actively restricted, and the selected loci are enriched for nucleic acidbased functions and include Argonaute-2 (AGO2) itself.
Keywords: Gene expression
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| Overall design |
Drosophila Schneider cells (S2) were treated with dsRNA against GFP for 8 days. The treatment was done in duplicate. Total RNA was extracted from the dsRNA treated cells using trizol.
Gene expression analysis was conducted using Drosophila Genome 2.0 Genechip® arrays (Affymetrix, Santa Clara, CA). Starting with 1ug of total RNA, biotin-labeled cRNA was produced using the Affymetrix 3¹ Amplification One-Cycle Target labeling kit according to manufacturer¹s protocol. For each array, 10ug of amplified cRNAs were fragmented and hybridized to the array for 16 hours in a rotating hybridization oven using the Affymetrix Eukaryotic Target Hybridization Controls and protocol. Slides were stained and washed as indicated in the Antibody Amplification Stain for Eukaryotic Targets protocol using the Affymetrix Fluidics Station FS450. Arrays were then scanned with an Affymetrix Scanner 3000 and data was obtained using the Genechip® Operating Software (Version 1.2.0.037).
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| Contributor(s) |
Okamura K, Balla S, Martin R, Liu N, Lai EC |
| Citation(s) |
18500351 |
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| Submission date |
May 07, 2008 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Nicolas Robine |
| Organization name |
Sloan-Kettering Institute
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| Department |
Developmental Biology
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| Lab |
Dr. Eric Lai's Lab
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| Street address |
430, East 67th Street, RRL 517
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platforms (1) |
| GPL1322 |
[Drosophila_2] Affymetrix Drosophila Genome 2.0 Array |
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| Samples (2) |
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| Relations |
| BioProject |
PRJNA106593 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE11370_RAW.tar |
19.1 Mb |
(http)(custom) |
TAR (of CEL, CHP) |
| Processed data included within Sample table |
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