Expression of SATs
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE2185 | GEO | 2005/03/31
SECONDARY ACCESSION(S): PRJNA91239
REPOSITORIES: GEO
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Project description:Expression of the sense-antisense transcripts (SATs). Methods: The sequences of 60mer DNA specific to the sense and antisense genes were chosen by K.K. DNAFORM (Japan), and Agilent Technologies manufactured custom oligo DNA microarray chips by using this information. RNA was labeled and hybridized using Fluorescent Direct Label Kits (oligo dT-primed labeling) (Agilent Technologies), according to the manufacturer's protocols. For labeling with random nanomers, we used the CyScribe First-Strand cDNA Labeling Kit (Amersham). The RNA samples used for microarray experiments were from mouse ES cells, SL10 cells (fibroblast cell line), brain, heart, and testis. The total RNA of brain, heart, and testis for array experiments was purchased from Ambion. The total RNA of ES and fibroblast cells was isolated using Trizol reagent (Invitrogen). The same total RNA samples were reciprocally labeled with Cy3 or Cy5, hybridized to the oligo DNA on the chip, and dye-normalized, and processed signals were obtained using Feature Extraction software (Agilent Technologies).
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Project description:We used BAF250b-/- ES cells, two independently derived clones, at 18 and 72 hr in culture and compared them with parental cell line (wild-type) at the same time in culture (two replications each). Total RNAs were extracted using Trizol (Invitrogen) according to manufacturer protocol. 2.5 ug of total RNA samples were labeled with Cy3-CTP using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent). A reference target (Cy5-CTP-labeled) was prepared from the Universal Mouse Reference RNA (Stratagene). Labeled targets were purified using an RNeasy Mini Kit (Qiagen) according to the Agilent's protocol, quantitated by a NanoDrop scanning spectrophotometer (NanoDrop Technologies), and hybridized to the NIA Mouse 44K Microarray v2.2 (whole genome 60-mer oligo; manufactured by Agilent Technologies, #014117) (Carter et al. 2005) according to the Agilent protocol (G4140-90030; Agilent 60-mer oligo microarray processing protocol - SSC Wash, v1.0). All hybridizations were carried out in the two color protocol by combining one Cy3-CTP-labeled experimental target and Cy5-CTP-labeled reference target. Microarrays were scanned on an Agilent DNA Microarray Scanner, using standard settings, including automatic PMT adjustment. Keywords: genetic modification design,time series design
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Project description:Total RNA was quantified by the NanoDrop ND-2000 (Thermo Scientific)and the RNA integrity was assessed using Agilent Bioanalyzer 2100 (Agilent Technologies). The sample labeling, microarray hybridization and washing were performed based on the manufacturer's standard protocols. Briefly, total RNA were transcribed to double strand cDNA, then synthesized into cRNA and labeled with Cyanine-3-CTP. The labeled cRNAs were hybridized onto the microarray. After washing, the arrays were scanned by the Agilent Scanner G2505C (Agilent Technologies).
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Project description:Increasing numbers of senseâantisense transcripts (SATs), which are transcribed from the same chromosomal location but in opposite directions, have been identified in various eukaryotic species, but the biological meanings of most SATs remain unclear. To improve understanding of natural senseâantisense transcription, we performed comparative expression profiling of SATs conserved among humans and mice. Using custom oligo-arrays loaded with probes that represented SATs with both protein-coding and non-proteinâcoding transcripts, we showed that 33% of the 291 conserved SATs displayed identical expression patterns in the two species. Among these SATs, expressional balance inversion of senseâantisense genes was mostly observed in testis at a tissue-specific manner. Northern analyses of the individual conserved SAT loci revealed that: (1) a smeary hybridization pattern was present in mice, but not in humans, and (2) small RNAs (about 60 to 80 nt) were detected from the exon-overlapping regions of SAT loci. In addition, further analyses showed marked alteration of senseâantisense expression balance throughout spermatogenesis in testis. These results suggest that conserved SAT loci are rich in potential regulatory roles that will help us understand this new class of transcripts underlying the mammalian genome. Keywords: Expression profile of mouse and human sense-antisense transcript The RNA samples used for the mouse oligo-array experiments came from the brain, NIH3T3 cells (fibroblast cell line), liver, heart, and testis. RNA from mouse tissue (C57BL/6J, 8 to 10 weeks, male and female mixed) and mouse fibroblast line NIH3T3 was isolated by using Trizol reagent (Invitrogen). The mouse custom oligo DNA microarray chip (microarray format: 11K) contained 3896 probes that represented genes in 1948 exon-overlapping SAT (sense-antisense transcript) pairs.The same total RNA samples were reciprocally labeled with Cy3 or Cy5, hybridized to the oligo DNA on the chip, and dye-normalized. The data on all genes on the chip were used to enable the Feature Extraction software to produce the processed signals. The total mean signal on the mouse chip in each hybridization experiment was adjusted with to a value of 4872.3. The RNA samples used for human microarray experiments came from brain, HF19 cells (fibroblast cell line), heart, liver and testis. The total brain, heart, and testis RNA used in the array experiments was purchased from Ambion. Total RNA was isolated from the fibroblast cells by using Trizol reagent (Invitrogen). The same total RNA samples were labeled with single color Cy3, hybridized to the oligo DNA on the chip, and dye-normalized; the processed signals were obtained by using Feature Extraction software (Agilent Technologies). The custom oligo DNA microarray chip (microarray format: 11K) contained 2793 probes for 1486 pairs of exon-overlapping SATs. The data on all genes on the chip were used to enable the Feature Extraction software to produce the processed signals. For further analysis, the Cy3-labeled processed signal was used as the processed signal from the expression of a particular gene. The total mean signal on the human chip in each hybridization experiment was adjusted to 1491.6, so that the relative differences in gene expression could be compared among cell lines and tissues.
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