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| Status |
Public on Oct 14, 2006 |
| Title |
Immunoprecipitation of PTB65 associated mRNAs |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
PTB is multifunctional RNA binding protein reported to be involved in splicing, 3' -end processing, stability and translational regulation.
Sample preparation methods (Gama-Carvalho et al, 2006, submitted)
Suspension HeLa cells were grown in DMEM, 10% FCS, Pen/Strep and split 1:2 the day before harvesting. Post-nuclear cytoplasmic extracts were obtained as described (Herbert and Hecht 1999), using 5x107 cells/ml of lysis buffer. mRNAs were isolated by immunoprecipitation from 200ul of precleared HeLa cell extract, using the anti-U2AF65 MC3 mAb (Gama-Carvalho, Krauss et al. 1997) or the Bb7 anti-PTB mAb (ATCC) for 2h at 4ºC. Immune complexes were precipitated with a slurry with 50% of protein A/protein G agarose beads (Amersham), blocked with 100ug/ul of tRNA and RNAse free BSA (Ambion), by rotating for 1h at 4ºC and washed with lysis buffer. Complexes bound to the beads were eluted with TES buffer (10mM Tris, 0.5M EDTA, 0.5% SDS, pH 8.0) by heating at 65ºC for 10 minutes and Trizol extracted. Polyadenilated RNA in the cell extract (input) and in the immunoprecipitation sample was reverse transcribed, end-tagged and PCR amplified using the Super SMART PCR cDNA synthesis kit from Clontech, following manufacturer instructions. 1ug of input or precipitated RNA was used per RT-PCR reaction to produce samples for microarray hybridization. For each set of samples (input and immunoprecipitated) a 100ul test PCR reaction was performed according to manufacturer instructions and analyzed by agarose gel electrophoresis to select ideal conditions for amplification in the linear range. PCR reactions were pooled to obtain enough material for microarray hybridization. 15ug of PCR amplified cDNA from input and immunoprecipitated samples were fragmented, end labeled with biotin and hybridized to Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays as described (Brodsky, Meyer et al. 2005), following standard Affymetrix hybridization protocols.
Brodsky, A. S., C. A. Meyer, et al. (2005). "Genomic mapping of RNA polymerase II reveals sites of co-transcriptional regulation in human cells." Genome Biol 6(8): R64.
Gama-Carvalho, M., R. D. Krauss, et al. (1997). "Targeting of U2AF65 to sites of active splicing in the nucleus." J Cell Biol 137(5): 975-87.
Herbert, T. P. and N. B. Hecht (1999). "The mouse Y-box protein, MSY2, is associated with a kinase on non-polysomal mouse testicular mRNAs." Nucleic Acids Res 27(7): 1747-53.
Keywords: RNA immunoprecipitation
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| Overall design |
We have used microarray hybridization coupled to RNA immunoprecipitation from HeLa cell cytoplasmic extracts using anti-PTB antibodies to identify mRNA molecules associated with the PTB splicing factor
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| Contributor(s) |
Gama-Carvalho M, Barbosa-Morais NL, Brodsky AS, Silver PA, Carmo-Fonseca M |
| Citation(s) |
17137510 |
| Submission date |
Oct 12, 2006 |
| Last update date |
Mar 25, 2019 |
| Contact name |
Margarida Gama-Carvalho |
| E-mail(s) |
m.gamacarvalho@fm.ul.pt
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| Organization name |
Institute of Molecular Medicine
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| Street address |
Av. Prof. Egas Moniz
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| City |
Lisboa |
| ZIP/Postal code |
1649-028 |
| Country |
Portugal |
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| Platforms (1) |
| GPL570 |
[HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array |
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| Samples (4)
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| GSM139832 |
Total cDNA from HeLa cell extract from Immunoprecipitation experiment 1 PTB |
| GSM139833 |
Immunoprecipitated cDNA HeLa cell extract from Immunoprecipitation experiment 1 PTB |
| GSM139834 |
Total cDNA from HeLa cell extract from Immunoprecipitation experiment 2 PTB |
| GSM139835 |
Immunoprecipitated cDNA HeLa cell extract from Immunoprecipitation experiment 2 PTB |
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| Relations |
| BioProject |
PRJNA97829 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE6021_RAW.tar |
35.3 Mb |
(http)(custom) |
TAR (of CEL) |
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