|
| Status |
Public on Nov 26, 2012 |
| Title |
GV with envelope RNA |
| Sample type |
RNA |
| |
|
| Source name |
GV with envelope RNA
|
| Organism |
Xenopus tropicalis |
| Characteristics |
gender: female
cell type: germinal vesicle (GV) with envelope
|
| Treatment protocol |
Oocytes were treated with collagenase with gentle swirling in plastic culture dishes for 2–3 hours to remove follicle cells. We developed a manual technique using jewelers’ forceps to remove the nuclear envelope without losing the GV contents. GVs were isolated in an isotonic saline solution at pH 5.6–5.8 (83 mM KCl, 17 mM NaCl, 6.0 mM Na2HPO4, 4.0 mM KH2PO4, 1 mM MgCl2, 1.0 mM dithiothreitol, adjusted to pH 5.6–5.8 with HCl). At this pH, the nuclear contents begin to gel within seconds after isolation from the oocyte. Simultaneously, the nuclear envelope swells away from the gelled contents and can be removed with jewelers’ forceps because it adheres to the forceps' tips. GV contents were collected in batches of 10–20 and transferred to 10 mM sodium citrate and 5mMEDTA (pH 5.0) for storage. For isolation of cytoplasmic RNA, oocytes were transferred from OR2 medium to the same solution used for GV isolation, except at pH 7.0. The GV was removed with jewelers’ forceps and the enucleated cytoplasm transferred in minimal liquid to a 2-mL Eppendorf tube on dry ice.
|
| Growth protocol |
Adult female X. tropicalis animals were anesthetized with 0.15% tricaine methane sulfo- nate, and one or both ovaries were removed surgically. Pieces of ovary were placed in OR2 medium (Wallace et al. 1973) at room temperature, where the oocytes maintain normal morphology and biochemical properties for several days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
TRIzol (Ambion) according to the manufacturer’s protocol.
|
| Label |
biotin
|
| Label protocol |
Following the Affymetrix ribosomal reduction step, 1 ug total RNA was combined with 2 ul T7 oligo(dT) primer and 2 ul Poly-A controls and brought to a volume of 12 ul. The samples were incubated at 70ºC for 10 minutes to label as per standard Affymetrix protocol.
|
| |
|
| Hybridization protocol |
The samples were ice quenched and combined with the hybridization cocktail (5 ul oligonucleotide B2 control, 15 ul 20X eukaryotic hybridization controls, 150 ul 2X hybridization buffer, 30 ul 100% DMSO and 70 ul water). After 10 minutes of pre-hybridizing the Xenopus tropicalis arrays at 45ºC, 60 rpm, 200 ul of cocktail was loaded onto each array and the arrays were hybridized for 16 hours at 45ºC, 60 rpm. The cocktail was removed and the arrays were stained and washed using the Affymetrix GeneChip Fluidics Station 450 and FS450_001 fluidics script.
|
| Scan protocol |
Fluorescence was detected using the Affymetrix G3000 GeneArray Scanner and image analysis of each GeneChip was perfomed through the Affymetrix GeneChip Command Console version 3.4 (AGCC v3.4) Affymetrix software.
|
| Description |
Total RNA from 716 hand-isolated germinal vesicles from oocytes of a mature Xenopus tropicalis frog
|
| Data processing |
Data were extracted from the CEL files, RMA normalized across all samples and converted to Log2 notation with Partek Genomics Suite (Partek, Missouri, USA). These values were labeled as to their source and the groups compared.
|
| |
|
| Submission date |
Oct 12, 2012 |
| Last update date |
Nov 26, 2012 |
| Contact name |
Joseph G. Gall |
| E-mail(s) |
[email protected]
|
| Organization name |
Carnegie Institution for Science
|
| Department |
Department of Embryology
|
| Street address |
3520 San Martin Drive
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21218 |
| Country |
USA |
| |
|
| Platform ID |
GPL10263 |
| Series (1) |
| GSE41530 |
Stable intronic sequence RNA (sisRNA), a new class of noncoding RNA from the oocyte nucleus of Xenopus tropicalis |
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