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Sample GSM182568 Query DataSets for GSM182568
Status Public on Aug 14, 2007
Title Otic vesicle at E10, replicate 1
Sample type RNA
 
Source name Mouse otic vesicle at E10
Organism Mus musculus
Characteristics Strain: CBA/J
Tissue: inner ear
Extracted molecule total RNA
Extraction protocol Inner ear dissections Timed pregnant CBA/J mice (Jackson Laboratory) were euthanized by placing in a chamber with CO2. While euthanasia was carried out by considering the morning of vaginal plug observation to be 0.5 days post-conception, the gestational stage of each embryo in a litter was determined individually by the number of somites (from E9 to E11) and various other developmental features such as the extent of digit and limb development, appearance of hair follicles and so on (beyond E11), as described by M.H. Kaufman in The Atlas of Mouse Development, revised edition, 1998, Academic Press, San Diego, CA. Embryos were removed and immediately placed in Hank’s Balanced Salt Solution (HBSS) supplemented with L-Glutamine on ice. All subsequent dissections were carried out in ice-cold Dulbecco’s calcium and magnesium-free phosphate buffered saline (PBS) solution. Whole inner ears, containing both inner ear (IE) and non-inner ear (NIE) tissues, were removed from embryos under a dissecting microscope (Nikon SMZ645, Boyce Scientific Inc) and transferred in 3-5mg/mL dispase (Gibco) in PBS on ice for either 8-10 minutes (gestational stages E9-E10.5) or 20-25 minutes (E11-E15) to facilitate the separation of IE epithelium from the surrounding NIE tissue. After incubation in dispase, the tissue was transferred to a 35mm glass bottom culture dish with a 10mm microwell (MatTek Corporation, MA) containing ice-cold PBS. IE epithelium was then micro-dissected under a stereo microscope (Nikon SMZ1500, Boyce Scientific Inc) using Dumostar 5 forceps (Biomedical Research Instruments) and immediately placed in TRIZOL (Invitrogen). For each gestational stage, two biological replicates were obtained, i.e., two pools of tissues of identical stage from different litters. From E9-E10, IE epithelia from 5-8 embryos were pooled per stage per replicate. From E10.5-E12, the ventral cochlear region and the dorsal vestibular region (without the endolymphatic duct) were separated and pooled separately for each stage, again from 5-8 embryos per stage per tissue-type per replicate. For stages E12.5-E15, the cochleae and the saccules were separately pooled, whereas the utricles and the ampullae of the three semi-circular canals were combined and pooled together (without the endolymphatic duct and canals) from 3-6 embryos per stage per tissue-type per replicate. The NIE tissue was also obtained from each stage and pooled followingly. From stage E9, the NIE tissue was obtained from 4-5 embryos per replicate. From E9.5-E10.5, the NIE tissue was obtained from 2-7 embryos per stage per replicate and then pooled together. From E11-E15, NIE tissue was obtained from 2-3 embryos per stage per replicate and then pooled. Hence, a total of 29 samples of IE and 3 of N-IE were obtained, each in duplicate, from 13 distinct developmental stages. Total RNA isolation, cDNA synthesis and amplification Total RNA was isolated from TRIZOL by following manufacturer’s instructions (Invitrogen) and resuspended in either 7-10ul (for stages E9-E10.5) or 15-20ul (for stages E11-E15) of distilled and deionized water (VWR). PolyA RNA was then amplified from at least one-fifth to one-half of total RNA using micro-cDNA methods described elsewhere [Hawkins RD et al. (2003) Human Molecular Genetics 12, 1261-1272] with the following modifications. Total RNA was denatured at 65oC in a 5ul volume in water for 5 minutes on a PCR machine (GeneAmp PCR system 9700, Applied Biosystems) and immediately added to an equal volume of oligo-(dT)24-coated paramagnetic beads (Dynal) in binding buffer. After 20 minutes at room remperature with periodic mixing, the supernatant was removed and beads washed twice with 50ul of wash buffer and once with 1X reverse transcriptase buffer (Invitrogen). All non-room temperature incubations were carried on the PCR machine mentioned above. The sequence of the 5’ SMART primer (referred to here as the Not1-SP6-3G primer) used was 5’-GCGGCCGCTATT TAGGTGACACTATAGAAGAGGG-3’ (synthesis by Invitrogen, PAGE-purified). Following cDNA synthesis, beads were washed twice with 50ul of water and once with 50ul of 1X Not1 digestion buffer (Invitrogen) prior to Not1 digestion at 37oC for 20 minutes in a 10ul reaction volume containing 15 units of enzyme in 1X digestion buffer. Post-digestion, beads were washed thrice with 50ul of water and subjected to 4 rounds of PCR cycles using the SP6 and T7-(T)24-VN primers whose sequences were 5’-ATTTAGGTGACACTATAGAA-3’ (synthesis by Invitrogen) and 5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG(T)24VN-3’ (synthesis by IDT, PAGE-purified), respectively. The PCR was carried out in a 50ul volume and included, in addition to the beads, 0.2mM dNTP mix, 0.02uM of each of the two primers, and 1X Advantage 2 Taq polymerase mix (BD Biosciences) in 1X Advantage 2 PCR buffer (40mM tricine-KOH pH 8.7, 15mM potassium acetate, 3.5mM magnesium acetate, 3.75ug/ml BSA, 0.005% tween-20, and 0.005% nonidet-P40). Cycle settings included an initial denaturation at 95oC for 2 minutes, then 95oC for 15 seconds, 42oC for 15 seconds, 68oC for 6 minutes, and an extra 4-minute elongation at 68oC for the last cycle. After this PCR, the supernatant was transferred to a new PCR tube to which was added a 50ul-mixture of 0.2mM dNTP mix, 0.2uM of the Not1-SP6-3G and T7-(T)24-VN primers each, and 1X Advantage 2 Taq polymerase mix in 1X Advantage 2 PCR buffer to obtain a total of 100ul reaction volume. This was subjected to additional 8 rounds of PCR cycles following the same settings as above except that primer annealing was carried out at 60oC. The resulting amplified cDNA was purified using a PCR purification kit (Qiagen) following the manufacturer’s instructions and eluted in 40ul of elution buffer.
Label Biotin
Label protocol To synthesize biotin-labeled target (cRNA) for hybridization to Affymetrix arrays, 22ul of the purified cDNA were used as template in in-vitro transcription reactions using the BioArray HighYield RNA Transcript Labeling Kit (ENZO Life Sciences, NY). Besides the template cDNA, all other components were added following the instructions provided by the manufacturer, and reactions were incubated at 37oC for 10 hours on the PCR machine mentioned above under 'extract protocol'. Labeled cRNA was purified using an RNA purification kit (Qiagen) following the manufacturer’s instructions and eluted in 50ul of water.
 
Hybridization protocol Fragmentation of cRNA was carried out following the standard Affymetrix procedure, and 10ug of fragmented cRNA from each sample was hybridized to the MOE 430A_2 gene chips for 16-18 hours at 45oC.
Scan protocol GeneChips were scanned using GeneChip Scanner 3000.
Description Pool of 15 otic vesicles at E10
Data processing The data were normalized and modeled using the PM/MM model in DNA Chip (dChip) program, June 2, 2005 version. All expression values below 10 were designated a default of 10. The Presence/Absence calls used were those generated by the Affymetrix MAS5 software.
 
Submission date Apr 17, 2007
Last update date Mar 18, 2009
Contact name Samin A Sajan
E-mail(s) sasajan@artsci.wustl.edu, lovett@genetics.wustl.edu
Organization name Washington University Medical School
Street address 4566 Scott Avenue
City Saint Louis
State/province MO
ZIP/Postal code 63110
Country USA
 
Platform ID GPL8321
Series (1)
GSE7536 Microarray expression profiling of discrete microdissected mouse inner ear tissues from E9 to E15 at half-day intervals.

Data table header descriptions
ID_REF
VALUE Normalized dChip expression/intensity value
ABS_CALL
STD_ERR

Data table
ID_REF VALUE ABS_CALL STD_ERR
1415670_at 1033.62 P 32.37
1415671_at 132.146 P 14.1201
1415672_at 704.224 P 26.4156
1415673_at 160.771 P 8.40931
1415674_a_at 652.401 P 16.0352
1415675_at 718.455 P 23.2342
1415676_a_at 2921.35 P 151.661
1415677_at 192.291 P 19.9768
1415678_at 1357.61 P 23.9994
1415679_at 1897.49 P 44.7091
1415680_at 483.615 P 15.7607
1415681_at 1757.21 P 33.0873
1415682_at 2899.43 P 58.2346
1415683_at 540.644 P 12.8558
1415684_at 140.607 P 10.3754
1415685_at 219.883 P 13.8176
1415686_at 435.932 P 10.9503
1415687_a_at 1297.7 P 44.9467
1415688_at 5078.34 P 134.561
1415689_s_at 211.651 P 6.01904

Total number of rows: 22626

Table truncated, full table size 634 Kbytes.




Supplementary file Size Download File type/resource
GSM182568.CEL.gz 3.3 Mb (ftp)(http) CEL
Processed data included within Sample table

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