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| Status |
Public on Jul 30, 2009 |
| Title |
HEK293 cells overexpressing KLHL29 intronic transcript (sense orientation), replicate number 4. |
| Sample type |
RNA |
| |
|
| Source name |
HEK293 cell line overexpressing KLHL29 intronic transcript
|
| Organism |
Homo sapiens |
| Characteristics |
HEK293 cell line obtained from ATCC (http://www.atcc.org/). Organ: Fetal kidney. Cell Type transformed with adenovirus 5 DNA.
|
| Biomaterial provider |
ATCC - American Type Culture Collection
|
| Treatment protocol |
Cells were transfected with the linearized pBudCE4.1 expression vector containing KLHL29 intronic S or AS insert, using FuGENE 6 Transfection Reagent (Roche Applied Science, Indianopolis, IN). After transfection, zeocin-resistant cells were selected at 50 µg/ml zeocin for one month. The heterogeneous population of selected transfectants was propagated, and cells were used for RNA extraction. Biological replicates overexpressing S or AS KLHL29 intronic transcript were produced by thawing and propagation of four frozen HEK293 cell populations, derived from the same transfection.
|
| Growth protocol |
Cells were cultured in DMEM, supplemented with 10% fetal bovine serum, in a humidified incubator with 5% CO2, at 37ºC.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using RNeasy kit (Qiagen, Hilden, Germany), treated with DNAse for 30 min, and quantified by measurement of absorption on a NanoDrop® ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE).
|
| Label |
Cy5
|
| Label protocol |
Cy5- or Cy3-labeled cRNA was obtained from 400 ng total RNA, amplified by in vitro transcription with T7-RNA polymerase, using the Agilent Low RNA Input Fluorescent Linear Amplification kit (Agilent Technologies). Two HEK293 replicates for each construct were labeled with Cy5, and another two with Cy3. In vitro synthesized control mRNA (Agilent RNA Spike-In kit) was spiked into the amplification and labeling assay.
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| |
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| Hybridization protocol |
Equal quantities of two cRNA samples (825 ng), derived from the same HEK293 transfection and labeled with Cy3 or Cy5, were mixed and hybridized to Whole Human Genome Oligo Microarray 4x44K (Agilent Technologies), strictly following the manufacturer protocol. Slides were washed and processed according to the Agilent 60-mer Oligo Microarray Processing protocol.
|
| Scan protocol |
RNA integrity was assessed using Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) prior to labeling procedures.
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| Description |
RNA integrity was assessed using Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) prior to labeling procedures.
|
| Data processing |
Data were extracted and pre-processed using Agilent Feature Extraction (v9.5) software. Genes were defined as expressed when their intensity signals were above the average negative control value plus three standard deviations; a set of 153 negative controls (Agilent Technologies) was present on the array. Only genes that were detected as expressed/not expressed in all four replicates of the same HEK293 transfection were used in further analysis. The intensity values between experiments were normalized by the 40% trimmed mean intensity. Measuring the coefficient of variance among the replicates, we have observed that one array experiment out of the four from each construction had a considerably higher number of outlier points, and the entire replicate experiment was excluded from the analyses.
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|
| Submission date |
Feb 02, 2009 |
| Last update date |
Mar 23, 2009 |
| Contact name |
Eduardo M Reis |
| E-mail(s) |
emreis@iq.usp.br
|
| Organization name |
Instituto de Química da Universidade de São Paulo
|
| Department |
Departamento de Bioquímica
|
| Street address |
Av. professor Lineu Prestes, 748
|
| City |
São Paulo |
| ZIP/Postal code |
05508-900 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE14681 |
Long intronic non-coding RNA from the KLHL29 locus regulates proliferation of human tumorigenic cell lines |
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