|
| Status |
Public on Jul 01, 2009 |
| Title |
E2-3 SMART cDNA |
| Sample type |
RNA |
| |
|
| Source name |
2-3 staged embryos
|
| Organism |
Drosophila melanogaster |
| Characteristics |
strain: Canton S wild type
developmental stage: 2-3 staged embryos
|
| Growth protocol |
D. melanogaster (Canton-S strain) flies were grown at 22 °C on standard cornmeal, molasses, agar and yeast medium. To obtain staged embryos, females were allowed to lay eggs for 2 h on 2% apple juice agar plates spread with live brewer's yeast. The plates were replaced several times, and finally 1-h embryos were collected, dechorionated and washed with Ringer Drosophila solution (182 mM KCl, 46 mM NaCl, 3 mM CaCl2, 10 mM Tris–Hcl, pH 7.2). Embryos were selected at stages 2–3 (syncytial blastoderm) or 5 (cellular blastoderm) and then allowed to continue their development in a humidified chamber at 25 °C. We hand-selected embryos at different developmental stages based on their morphological characters (Campos-Ortega and Hartenstein, 1985) and rapidly froze them in liquid N2. Embryos were kept at −80 °C for 1–2 weeks.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Embryos were collected at 5 developmental intervals (stages 2-3, 5, 6-7, 8-9 and 10-12, according to Campos-Ortega & Harstenstein, 1985). Total RNA was extracted from staged embryos (n=100) using the RNAWIZ reagent (Ambion). Embryos were carefully homogenized in a 1.5 mL eppendorf tube with 1 mL of RNAWIZ reagent using a plastic tissue grinder. To improve RNA yield, the homogenized was passed throughout Qiashreder columns (Qiagen) by centrifugation at room temperature for 2 min at 10,000 x g. The quantity and quality of the RNA were assessed by OD260/280 and by electrophoresis on a 1.2% formaldehyde-agarose gel. Typical yield was 0.18-0.23 µg RNA/embryo. Total RNA was used to synthesize double strand cDNA using SMART cDNA Synthesis kit (Clontech) according to manufacturer´s indications.
|
| Label |
32P
|
| Label protocol |
The 32P-labeled probes were prepared from SMART-cDNAs by incorporation of [α-32P]dCTP using the Random Primers DNA Labelling System (Invitrogen), according to manufacturer´s indications. Unincorporated radioactive nucleotides were removed using the QIAquick Nucleotide Removal (Qiagen).
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| |
|
| Hybridization protocol |
The labeled cDNA products were denatured and immediately used for membrane hybridization. Hybridization conditions were as follows: pre-hybridization in 5 ml of hybridization buffer (5X SSC, 5X Denhardt solution, 1% SDS, and 50% formamide) for 1–3 h at 42 ºC. Hybridization was performed in the same buffer for 16–18 h at 42 ºC. Membranes were washed at room temperature with two 10-min changes of 2× SSC and 2× SSC plus 0.1% SDS, then with two 15-min changes of 1× SSC plus 0.1% SDS at 60 °C and finally with two 15-min changes of 0.1× SSC.
|
| Scan protocol |
Membranes were sealed in plastic bags and placed in Imaging Screen-K (Biorad) for 12-24 hours. Radioactive images were obtained using a scanner Personal Molecular Image FX (BioRad) at 50 µm/pixel resolution. Intensities values were measured using VersArray Analizer v.4.5.1.46 (BioRad).
|
| Description |
SMART cDNA was synthesized from total RNA from 2-3 staged embryos, labeled with 32P in a random primers labeling method.
|
| Data processing |
Raw values were measured as the volume of pixels within a circle encompassing the spot. Local background values were measured in the corners of spot and were subtracted from the signal intensity values for each spot. Pearson´s correlation coefficient was used to establish the quality of replicated membranes. Spots that showed: (1) Signal Mean < (Background Mean + (1 x Background Standard Deviation); (2) coefficient of variation (CV) > 0.5 between duplicate spots within membranes (Dudoit et al. 2000) or (3) qcom < 0.8 (Wang et al. 2001) were considered as low-quality spots and were removed. After data filtering, net intensity values were normalized against Dap spike mRNA (membranes A, B and C) or RP49 housekeeping gene (membrane D) intensity values. When genes were represented by more than one clone, mean intensity values were calculated.
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| |
|
| Submission date |
Feb 25, 2009 |
| Last update date |
Feb 25, 2009 |
| Contact name |
Alejandro Zúñiga |
| E-mail(s) |
jano@inta.cl
|
| Organization name |
INTA - Universidad de Chile
|
| Lab |
Bioinformática y Expresion Génica
|
| Street address |
Macul 5540
|
| City |
Santiago |
| ZIP/Postal code |
7810000 |
| Country |
Chile |
| |
|
| Platform ID |
GPL8228 |
| Series (1) |
| GSE15000 |
Subtractive Hybridization Reveals Novel Genes Associated with D. melanogaster Early Embryogenesis. |
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