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| Status |
Public on Dec 19, 2012 |
| Title |
RIP: miR-202 mimic-transfected HeLa cells vs. negative control |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
Potential miR-202 targets were identified using a targetome-wide RIP-based microarray. HeLa cells were transfected with either a miR-202 mimic or a scrambled single-stranded RNA negative control. RNA was isolated from total cell lysate prior to IP and from antibody-immobilized Protein G agarose beads-RNP complexes (post-IP).
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| Overall design |
Ribonucleoprotein IP was performed using the RIP-Assay kit for microRNA (MBL) according to the protocol described by the manufacturer. Briefly, anti-EIF2C2/Ago2 monoclonal antibody (Novus Biologicals, LLC) was incubated with Protein G plus agarose beads (Pierce) at 4°C overnight to prepare antibody-immobilized beads. 20 million cells were harvested and washed four times with ice-cold DEPC-treated PBS. The cell pellet was lysed with 500 μl of lysis buffer and the supernatant was incubated with Protein G agarose beads without antibody to reduce nonspecific adsorption. The cell lysate was then transferred into a tube containing antibody-immobilized Protein G agarose beads and incubated for 3 hours at 4°C. Genome-wide expression levels were analyzed in total RNA samples from total cell lysate and antibody-immobilized Protein G agarose beads-RNP complexes from cells transfected with miR-202 mimic or negative control using the Agilent 44K 60-mer human whole-genome microarray. Signal hybridization and scans were performed by MOGene, LC (St Louis, MO) (run in biological duplicate). The normalized signal intensities of probes from RNP-bead complexes (post-IP fraction) were divided by the signal intensities from total cell lysate (pre-IP fraction) in miR-202 mimic-transfected cells. Transcripts were identified as members of the global miRNA targetome if they exhibited an enrichment fold change > 2.0. From this list, post-IP to pre-IP signal intensity ratios in miR-202 mimic-transfected cells were divided by post-IP to pre-IP signal intensity ratios in NC cells. Transcripts exhibiting normalized signal ratios of > 1.5 were considered to be bound with miR-202 in the RNA-induced silencing complex (RISC), and therefore potential direct miR-202 targets.
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| Contributor(s) |
Zhu Y, Hoffman AE, Liu R, Fu A, Zheng T, Slack F |
| Citation(s) |
23334589 |
| Submission date |
Dec 18, 2012 |
| Last update date |
Jan 23, 2019 |
| Contact name |
Alan Fu |
| E-mail(s) |
alan.fu@yale.edu
|
| Organization name |
Yale University
|
| Street address |
60 College St.
|
| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06510 |
| Country |
USA |
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| Platforms (1) |
| GPL6480 |
Agilent-014850 Whole Human Genome Microarray 4x44K G4112F (Probe Name version) |
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| Samples (4)
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| GSM1054385 |
Post-IP miR-202 mimic-transfected vs. Pre-IP miR-202 mimic-transfected replicate 1 |
| GSM1054386 |
Post-IP miR-202 mimic-transfected vs. Pre-IP miR-202 mimic-transfected replicate 2 |
| GSM1054387 |
Post-IP scrambled NC-transfected vs. Pre-IP scrambled NC-transfected replicate 1 |
| GSM1054388 |
Post-IP scrambled NC-transfected vs. Pre-IP scrambled NC-transfected replicate 2 |
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| Relations |
| BioProject |
PRJNA184015 |
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