|
| Status |
Public on Dec 05, 2011 |
| Title |
IGFBP2 silencing - experiment 1A |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
RH36 treated with siRNA pool for IGFBP2 target gene (test)
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: rhabdomyosarcoma cell line
cell line: RH36
sirna transfection: IGFBP2 transcript was silenced with siRNA pool (Darmachon)
|
| Biomaterial provider |
Lucia Tombolan (Dept. Biology and CRIBI - University of Padova)
|
| Treatment protocol |
RH36 cells at 50-70% confluent status were transfected with pool siRNA for target gene IGFBP2 (siIGFBP2) using Dharmafect 3 transfection reagent following the manufacturer’s instructions (Dharmacon, Thermo Scientific, Lafayette, CO). After 48 hours from transfection the cells were harvested with TRIzol (Invitrogen) for total RNA extraction. The efficacy of the knockdown of the gene was evaluated at mRNA or protein level by RT-PCR and western blot.
|
| Growth protocol |
DMEM + 10% Fetal Calf Serum (FCS).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with TRIzol method (Invitrogen). The procedure was:
1. Addition of chloroform 0.2ml/1ml Trizol and shake for 1 minute;
2. Leave in ice for 15 minutes and then centrifuge at 4º C for 20 minutes at 14,000 X g;
3 Transfer the supernatant to 1.5 ml microfuge non-stick tubes and add an equal volume of isopropanol to precipitate the RNA;
4. Incubate at 4º C for 20 minutes and centrifuge at 4º C for 20 minutes at 14,000 X g;
5. Discard the supernatant and wash with ethanol 75% centrifuging for 15 minutes at 4º C at 14,000 X g;
6. Pellet was resuspended in ultraPURE™ distilled water DNase, RNase Free (Gibco).
|
| Label |
Cy5
|
| Label protocol |
Label protocol Extracted total RNA was quantized by UV adsorption in a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) and analyzed for quality by capillary electrophoresis in an Agilent 2100 Bioanalyzer. 1ug of total RNA for each of three siRNA experiments (siIGFBP2-1,2,3) was linearly amplified with with the MessageAmp™ aRNA amplification kit (Ambion). 5ug of the amplified RNA (aRNA) have been dried for 12 hours using a Heto Lyolab 3000 freeze-dryer (Heto-Holten A/S) and coupled with Cy5 post-labeling dye (Amersham, GE Healthcare). Coupling was performed for 45 minutes at room temperature. Unincorporated dyes were eliminated using MEGAclear™ Kit (Ambion). Labeled aRNA was eluted in ultraPURE™ distilled water DNase, RNase Free (Gibco) and quantized in a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific).
|
| |
|
| Channel 2 |
| Source name |
RH36 treated with siRNA pool for non-targeting gene (Reference)
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: rhabdomyosarcoma cell line
cell line: RH36
sirna transfection: treated with siRNA pool for non-targeting gene (Dharmacon)
|
| Biomaterial provider |
Lucia Tombolan (Dept. Biology and CRIBI - University of Padova)
|
| Treatment protocol |
RH36 cells at 50-70% confluent status were transfected with non-targeting siRNA pool (siCONTROL) using Dharmafect 3 transfection reagent following the manufacturer’s instructions (Dharmacon, Thermo Scientific, Lafayette, CO). After 48 hours from transfection the cells were harvested with TRIzol (Invitrogen) for total RNA extraction. The efficacy of the knockdown of the gene was evaluated at mRNA or protein level by RT-PCR and western blot.
|
| Growth protocol |
DMEM + 10% Fetal Calf Serum (FCS).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with TRIzol method (Invitrogen). The procedure was:
1. Addition of chloroform 0.2ml/1ml Trizol and shake for 1 minute;
2. Leave in ice for 15 minutes and then centrifuge at 4º C for 20 minutes at 14,000 X g;
3 Transfer the supernatant to 1.5 ml microfuge non-stick tubes and add an equal volume of isopropanol to precipitate the RNA;
4. Incubate at 4º C for 20 minutes and centrifuge at 4º C for 20 minutes at 14,000 X g;
5. Discard the supernatant and wash with ethanol 75% centrifuging for 15 minutes at 4º C at 14,000 X g;
6. Pellet was resuspended in ultraPURE™ distilled water DNase, RNase Free (Gibco).
|
| Label |
Cy3
|
| Label protocol |
Label protocol Extracted total RNA was quantized by UV adsorption in a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) and analyzed for quality by capillary electrophoresis in an Agilent 2100 Bioanalyzer. 1ug of total RNA for pool of three siRNA experiments with non-targeting siRNA was linearly amplified with with the MessageAmp™ aRNA amplification kit (Ambion). 5ug of the amplified RNA (aRNA) have been dried for 12 hours using a Heto Lyolab 3000 freeze-dryer (Heto-Holten A/S) and coupled with Cy5 post-labeling dye (Amersham, GE Healthcare). Coupling was performed for 45 minutes at room temperature. Unincorporated dyes were eliminated using MEGAclear™ Kit (Ambion). Labeled aRNA was eluted in ultraPURE™ distilled water DNase, RNase Free (Gibco) and quantized in a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific).
|
| |
|
| |
| Hybridization protocol |
Ranges along 350 and 300 pico-moles and 2.7 and 3.0 µg of aRNA labeled with Cy3 and Cy5 were co-precipitated with the ammonium acetate and ethanol. Pellet was resuspended in 110 µl with the hybridization solution composed by SSC 5X, SDS 0.1%, Formamide 25% and salmon sperm purified DNA 100 ng/µl and carried out in an automatic hybridization station (ArrayBooster, Advalytix) at 48° C for 30 hours in the competitive hybridization. Microarray slides, before hybridization, have been blocked with the pre-hybridization solution (SSC 5X, SDS 0.1%, salmon sperm purified DNA 100 ng/ul and Denhardt's solution 5X) for 6 hours at 48° C. After the hybridization slides were washed at room temperature with 1X SSC 0.2% SDS for 4 minutes one time; 0.1X SSC 0.2% SDS for 4 minutes one time; 0.2X SSC for 3 minutes two times each with new solution and 0.1X SSC for 3 minutes one time.
Slides were dried centrifuging 1 minute at 800 X g at 20° C.
|
| Scan protocol |
After stringent washings, fluorescence left in the microarray slides were read with the ScanArray LITE confocal laser scanner (PerkinElmer) with 5 µm resolution. The scans images were quantified with ScanArray Express (PerkinElmer) software using the fixed circle method.
|
| Description |
We compared the transcription profiles of IGFBP2 silencing cells (RH36) by microarray competitive hybridization against siCONTROL cells.
|
| Data processing |
Fluorescence intensity was determined with the ScanArray Express software (PerkinElmer) using fixed circle method and median spot intensity background subtracted was normalized with MIDAS tool (from TIGR TM4 suite; Saeed et al., 2003) using sequentially global mean normalization and local mean normalization (LOWESS) methods. Lowess normalization was performed without distinguishing between different sub-arrays.
From this analysis were included only the array positions containing a 70-mer oligonucleotides. The generated normalized matrix was loaded in to TIGR MeV software.
|
| |
|
| Submission date |
Jul 27, 2010 |
| Last update date |
Dec 05, 2011 |
| Contact name |
Gerolamo Lanfranchi |
| E-mail(s) |
stefano.cagnin@unipd.it
|
| Phone |
+39-0498276219
|
| Organization name |
University of Padova
|
| Department |
CRIBI - Biotechnology Center and Biology Department
|
| Lab |
Functional Genomics Lab
|
| Street address |
Via U. Bassi, 58/B
|
| City |
Padova |
| ZIP/Postal code |
35131 |
| Country |
Italy |
| |
|
| Platform ID |
GPL2136 |
| Series (1) |
| GSE23196 |
High IGFBP2 Expression Correlates with Tumor Severity in Pediatric Rhabdomyosarcoma. |
|
