Comment[ArrayExpressAccession] E-GEOD-30440 MAGE-TAB Version 1.1 Public Release Date 2013-04-24 Investigation Title Toward synaptic transcriptomes: Direct sequencing and identification of RNAs actively transported by the kinesin complex from the cell body to synapses in Aplysia neurons Comment[Submitted Name] Toward synaptic transcriptomes: Direct sequencing and identification of RNAs actively transported by the kinesin complex from the cell body to synapses in Aplysia neurons Experiment Description Specific mRNAs are transported from the cell body to synapses where their translation can modify communication of pre-existing synapses and induce formation of new synaptic connections in response to learning. Little is known, however, about the identity of the RNAs that are actively transported and when and how these RNAs are utilized during learning. By focusing on RNAs that are associated with kinesin, a motor protein that transports gene products from the cell body to synapses, we have now applied microarrays and 454 sequencing to identify actively transported RNAs from the Aplysia central nervous system. Using a library prepared from the kinesin complex immunoprecipitated from the central nervous system (CNS), we have identified thousands of unique transcripts, of which ~600 mRNAs were annotated. Two sample comparison: kinesin IP vs. control. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Kohn Puthanveettil Kohn Kandel Moroz Person First Name Andrea Sathyanarayanan Andrea Eric Leonid Person Mid Initials B V B R L Person Email abkohn@msn.com Person Affiliation University of Florida Person Phone 904 461-4007 Person Fax 904 461-4052 Person Address Whitney lab, University of Florida, 9505 Ocean Shore Blvd, St Augustine , FL, USA Person Roles submitter Protocol Name P-GSE30440-1 P-GSE30440-5 P-GSE30440-6 P-GSE30440-2 P-GSE30440-3 P-GSE30440-4 P-GSE30440-7 Protocol Description The balancing between the Cy3 and Cy5 signals within each array was performed by the RANK order method using the LOWESS smoothing procedure as implemented in the Agilent Feature Extraction software version 7.0. The background for the Cy3 and Cy5 channels on all arrays analyzed 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 the Cy3 and Cy5 channels from each array was used for further analysis (M-bM-^@M-^\floorM-bM-^@M-^]). 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. ID_REF = VALUE = Lowess-normalized log10 ratio of green processed signal/red processed signal FoldChange = Antilog of LogRatio LogRatioError = Error of LogRatio PValueLogRatio = P-Value LogRatio gProcessedSignal = Green ProcessedSignal rProcessedSignal = Red ProcessedSignal gProcessedSigError = Green ProcessedSigError rProcessedSigError = Red ProcessedSigError LogRatio = Lowess-normalized log10 ratio of red processed signal/green processed signal cRNA production was performed using AgilentM-bM-^@M-^Ys Low RNA Input Linear Amplification kit (Agilent Technologies). All amplification reactions involved only one round of amplification. The concentration of the cRNA was determined by GeneSpec III and the 2100 Bioanalyzer. The cRNA was labeled either using the direct method in AgilentM-bM-^@M-^Ys Low RNA Input Linear Amplification kit or with an indirect technique using the MicroMax ASAP RNA labeling kit (PerkinElmer). The recommended amount of 750 ng of labeled cRNA was fragmented and hybridized to the arrays using the recommended procedures described in the In situ Hybridization Kit Plus (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 manufacturer's recommended 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 mechanically removed with fine forceps and scissors, and cells were isolated. Neurons were then fixed in 75% ethanol for several minutes. 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 15-17M-: C. Isolation of an RNA transport complex from the Aplysia central nervous system: Custom antibodies to Aplysia Kinesin were used in a pull-down experiment. mRNAs associated with the kinesin complex were extracted using RNAqueous or RNAqueous-Micro (Ambion, Austin, TX) kits depending on sample size. Before further processing of the RNA, quality was checked using a 2100 Bioanalyzer (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 system (Mirai Bio). The arrays were scanned on an Agilent Technologies G2565AA Microarray Scanner. Data were extracted after scanning using AgilentM-bM-^@M-^Ys Feature Extraction 5.1.1 software. Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name ANTIBODY Experimental Factor Type antibody Comment[SecondaryAccession] GSE30440 Comment[GEOReleaseDate] 2013-04-24 Comment[ArrayExpressSubmissionDate] 2011-07-06 Comment[GEOLastUpdateDate] 2013-04-25 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-30440.sdrf.txt