|
| Status |
Public on Jan 05, 2007 |
| Title |
L7 vs sensory cluster 2 (DAA array) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
L7 Neuron
|
| Organism |
Aplysia californica |
| Characteristics |
Aplysia californica weighing 100-160g
L7 was identified electorphysiologically
|
| Biomaterial provider |
Leonid L Moroz laboratory
|
| Treatment protocol |
Aplysia californica weighing 100-160g were used. Animals were anesthetized by injection of 60% (volume/body weight) isotonic MgCl2 (337mM) prior to removal of the CNS. Ganglia were mechanical removed with fine forceps and scissors and cell were isolated as described above. Neurons were then fixed in 75% ethanol for several minutes and RNA isolated immediately.
|
| Growth protocol |
Aplysia californica were obtained from the NIH/University of Miami National Resource for Aplysia and held in 40-400 liter aquaria with circulating fresh seawater at 15-17º C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using RNAqueousTM or RNAqueous-MicroTM (Ambion, Austin, TX) kits depending on sample size. Before further processing of the RNA, quality was checked using a 2100 BioanalyzerTM (Agilent Technologies). Small aliquots of extracted RNA were loaded on a 6000 Nano Lab chip that produces an electropherogram image along with a gel-like image of the sample representing peak ratios of the 28s/18s RNA contained in the sample. Moreover it provides information about potential DNA contamination and the approximate RNA concentration. Additionally RNA concentration was measured spectrophotometrically by using the GeneSpec III systemTM (Mirai Bio).
|
| Label |
Cy3
|
| Label protocol |
cRNA production was preformed using Agilent’s Low RNA Input Linear Amplification kitTM (Agilent Technologies). All amplification reactions involved only one round of amplification. Concentration of the cRNA was determined by GeneSpec IIITM and the 2100 Bioanalyzer both described above. The cRNA was labeled either using the direct method in Agilent’s Low RNA Input Linear Amplification kit or with an indirect technique using the MicroMax ASAP RNA labeling kitTM (PerkinElmer).
|
| |
|
| Channel 2 |
| Source name |
Sensory cluster
|
| Organism |
Aplysia californica |
| Characteristics |
Aplysia californica weighing 100-160g
Sensory cluster was identified and isolated
|
| Biomaterial provider |
Leonid L Moroz laboratory
|
| Treatment protocol |
Aplysia californica weighing 100-160g were used. Animals were anesthetized by injection of 60% (volume/body weight) isotonic MgCl2 (337mM) prior to removal of the CNS. Ganglia were mechanical removed with fine forceps and scissors and cell were isolated as described above. Neurons were then fixed in 75% ethanol for several minutes and RNA isolated immediately.
|
| Growth protocol |
Aplysia californica were obtained from the NIH/University of Miami National Resource for Aplysia and held in 40-400 liter aquaria with circulating fresh seawater at 15-17º C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using RNAqueousTM or RNAqueous-MicroTM (Ambion, Austin, TX) kits depending on sample size. Before further processing of the RNA, quality was checked using a 2100 BioanalyzerTM (Agilent Technologies). Small aliquots of extracted RNA were loaded on a 6000 Nano Lab chip that produces an electropherogram image along with a gel-like image of the sample representing peak ratios of the 28s/18s RNA contained in the sample. Moreover it provides information about potential DNA contamination and the approximate RNA concentration. Additionally RNA concentration was measured spectrophotometrically by using the GeneSpec III systemTM (Mirai Bio).
|
| Label |
Cy5
|
| Label protocol |
cRNA production was preformed using Agilent’s Low RNA Input Linear Amplification kitTM (Agilent Technologies). All amplification reactions involved only one round of amplification. Concentration of the cRNA was determined by GeneSpec IIITM and the 2100 Bioanalyzer both described above. The cRNA was labeled either using the direct method in Agilent’s Low RNA Input Linear Amplification kit or with an indirect technique using the MicroMax ASAP RNA labeling kitTM (PerkinElmer).
|
| |
|
| |
| Hybridization protocol |
The recommended amount of 750 ng of labeled cRNA was fragmented and hybridized to the arrays using recommended procedures described in the In situ Hybridization Kit PlusTM (Agilent Technologies). Controls provided in the In situ Hybridization Kit were spiked in at the time of hybridization. The arrays were washed according to the manufactures recommended protocol.
|
| Scan protocol |
The arrays were scanned on an Agilent Technologies G2565AA Microarray ScannerTM. Data were extracted after scanning using Agilent’s Feature Extraction 5.1.1TM software
|
| Description |
L7 neuron vs sensory cluster
|
| Data processing |
The balancing between the Cy3 and Cy5 signals within each array was performed by the RANK order method using LOWESS smoothing procedure as implemented in the Agilent feature extraction software version 7.0 (http://chem.agilent.com/scripts/LiteraturePDF.asp?iWHID=30997). The background on all arrays analyzed for the Cy3 and Cy5 channels was observed to be nearly identical (41.8 1.1) over all 44,000 features. No local background subtraction was performed; instead the data was filtered by P-values obtained from fitting the signal and background values and spatial detrending algorithm. Only data that had P-values of less than or equal to 0.01 (meaning feature signal is significantly and positively higher than background signal) on both Cy3 and Cy5 channels from each array was used for further analysis (“floor”). A suite of algorithms are also included in the processing of the final TIFF image involving an error-model proprietary to Agilent that has been pre-determined to give the best corrected data for downstream analysis. All experiments were performed in triplicates from three independent samples from 3 different animals.
|
| |
|
| Submission date |
Apr 06, 2006 |
| Last update date |
Jan 05, 2007 |
| Contact name |
Andrea B Kohn |
| E-mail(s) |
abkohn@msn.com
|
| Phone |
904 461-4007
|
| Fax |
904 461-4052
|
| Organization name |
University of Florida
|
| Department |
Whitney lab
|
| Lab |
Leonid L Moroz
|
| Street address |
9505 Ocean Shore Blvd
|
| City |
St Augustine |
| State/province |
FL |
| ZIP/Postal code |
32080 |
| Country |
USA |
| |
|
| Platform ID |
GPL3636 |
| Series (2) |
| GSE4626 |
L7 vs sensory cluster (Discovery array DAA) |
| GSE4628 |
The Neuronal Transcriptome of Aplysia californica: A Platform for the Neurogenomics of Defined Neurons |
|
