|
| Status |
Public on Dec 15, 2010 |
| Title |
D.rerio_GFP+ cells vs GFP- cells from 48hpf embryos tails_2 Replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
D.rerio_GFP-cells from 36hpf embryo tails_2 rep2
|
| Organism |
Danio rerio |
| Characteristics |
developmental stage: 48 hpf zebrafish embryo
cell type: GFP- cell from embryonic tails
genotype: Cldnb:gfp transgenic
|
| Treatment protocol |
We used the tails of the Cldnb:gfp transgenic embryos in order to avoid recovering GFP-expressing cells in tissues other than the lateral line. Fish were anaesthetized and the tail was transected with a sharp scalpel adjacent to the end of the yolk extension. Tails of embryos were recovered from 36 hpf (for isolating primordium cells) or from 48 hpf (for neuromast cells) embryos, then transferred to Hank's medium containing 0.25% trypsin and 1mM EDTA and incubated for 15 to 30 min at room temperature during which they were dissociated mechanically by pipetting them up and down every 5 minutes. The digestion was stopped by adding fetal bovine serum to 10% and cell suspensions were then filtered through 40 µm nylon mesh, washed twice with PBS, pelleted by centrifugation at 600Xg for 2 minutes and resuspended in L-15 medium supplemented with 10% fetal bovine serum. FACS of single cell suspensions (GFP+ cell fraction and GFP- cell fraction) was performed at room temperature using a FACSAria.
|
| Growth protocol |
Embryos were grown at 28.5°C in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, and 0.1% Methylene Blue)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
Approximately 500 ng to 800 ng RNA was linearly amplified by using the Amino Allyl MessageAmp II aRNA Amplification kit (Ambion) with yields ranging from 12 to 30 µg of aRNA. aRNA samples were split and labeled, half with Cy3 mono NHS ester and half with Cy5 mono NHS ester (CyDyes from GE Healthcare; post-labeling reagents from the MessageAmp II kit).
|
| |
|
| Channel 2 |
| Source name |
D.rerio_GFP+cells from 36hpf embryo tails_2 rep2
|
| Organism |
Danio rerio |
| Characteristics |
developmental stage: 48 hpf zebrafish embryo
cell type: GFP+ cell from embryonic tails
genotype: Cldnb:gfp transgenic
|
| Treatment protocol |
We used the tails of the Cldnb:gfp transgenic embryos in order to avoid recovering GFP-expressing cells in tissues other than the lateral line. Fish were anaesthetized and the tail was transected with a sharp scalpel adjacent to the end of the yolk extension. Tails of embryos were recovered from 36 hpf (for isolating primordium cells) or from 48 hpf (for neuromast cells) embryos, then transferred to Hank's medium containing 0.25% trypsin and 1mM EDTA and incubated for 15 to 30 min at room temperature during which they were dissociated mechanically by pipetting them up and down every 5 minutes. The digestion was stopped by adding fetal bovine serum to 10% and cell suspensions were then filtered through 40 µm nylon mesh, washed twice with PBS, pelleted by centrifugation at 600Xg for 2 minutes and resuspended in L-15 medium supplemented with 10% fetal bovine serum. FACS of single cell suspensions (GFP+ cell fraction and GFP- cell fraction) was performed at room temperature using a FACSAria.
|
| Growth protocol |
Embryos were grown at 28.5°C in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, and 0.1% Methylene Blue)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
Approximately 500 ng to 800 ng RNA was linearly amplified by using the Amino Allyl MessageAmp II aRNA Amplification kit (Ambion) with yields ranging from 12 to 30 µg of aRNA. aRNA samples were split and labeled, half with Cy3 mono NHS ester and half with Cy5 mono NHS ester (CyDyes from GE Healthcare; post-labeling reagents from the MessageAmp II kit).
|
| |
|
| |
| Hybridization protocol |
200 picomoles of Cy3 and 100 picomoles of Cy5 labeled cRNAs are put in a total volume of 24 ul (qsp with DEPC H20) and 6 microliter of 5x fragmentation buffer from Affymetrix. the mixture is incubated at 94 ¡C for 15 minutes. Then, 25 ul of 2X hybridization buffer (250 _l Formamide, 250 _l 20xSSC and 20 _l 10%SDS). The mixture is then denatured for 2 minutes and applied on the Maui (BioMicro Systems, Inc) hybridization chamber/ array slide sandwich. The hybridization is performed at 45 degre for 20 hours.
|
| Scan protocol |
Microarray slides were scanned with a confocal laser (Agilent Technologies, Palo Alto, CA)
|
| Data processing |
PLab/DeArray. Data points with quality values below 0.7 were eliminated and the datasets were Lowess normalized. Normalized data were log-transformed and GFP+ and GFP- values were separately averaged over the eight experiments. Fold differences were calculated from log averages and Student’s t-test with Benjamini and Hochberg correction to generate p-values that were used to determined statistical significance. FileMaker Pro 9 (FileMaker, Inc.) and GeneSifter (http://www.genesifter.net/) softwares were used.
|
| |
|
| Submission date |
Nov 26, 2010 |
| Last update date |
Dec 15, 2010 |
| Contact name |
Viviana Gallardo |
| E-mail(s) |
gallardov@mail.nih.gov
|
| Phone |
301-594-9221
|
| Organization name |
NHGRI/NIH
|
| Street address |
50 South Dr. Bldg 50. Room 5535
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL4021 |
| Series (1) |
| GSE25617 |
Molecular dissection of the migrating posterior lateral line primordium during early development in zebrafish |
|
