|
| Status |
Public on Dec 17, 2015 |
| Title |
BCPAP miR-146b-5p mimic transfected |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
BCPAP miR-146b-5p mimic transfected
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Human Papillary thyroid cancer cell line, BCPAP
|
| Treatment protocol |
The cells were seeded (3 X 105 cells per well in 6-well plate) in antibiotic-free medium for 24 hours prior to transfection. DharmaFECT transfection reagent (Dharmacon RNA Technologies, Lafayette, CO) was used to transfect cells with mimic miR-146b (DharmaconRNATechnologies) for 24 hours at 50% confluence following the manufacturers instructions. The miRIDIAN miRNA mimic negative control (Dharmacon RNA Technologies) was used as control. Samples were collected after 24 hours of miRNA mimic transfections for quantification of miRNA. in antibiotic-free medium for 24 hours prior to transfection. DharmaFECT transfection reagent (Dharmacon RNA Technologies, Lafayette, CO) was used to transfect cells with mimicmiR- 146b (DharmaconRNATechnologies) for 24 hours at50% confluence following the manufacturer’s instructions. The miRIDIAN miRNA mimic negative control (Dharmacon RNA Technologies) was used as control. Samples were collected after 24 hours of miRNA mimic transfections for quantification of miRNA.
|
| Growth protocol |
The human PAPILLARY thyroid cancer cell line BCPAP was routinely cultured in RPMI 1640 containing 10% FBS, 100 U/mL penicillin, 100 g/mL streptomycin, and 2mML-glutamine(GIBCO,Rockville, MD) at 37°C in a humidified chamber containing 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from separated cell populations using Qiagen RNeasy Mini Kit (QIAGEN, Valencia, CA)
|
| Label |
Cy5
|
| Label protocol |
RNA from BCPAP control was labeled by Cy3 and RNA from BCPAP miR-146b-5p mimic transfected was labeled by Cy5. 0.2 μg of total RNA was amplified by a Low Input Quick-Amp Labeling kit (Agilent Technologies, USA) and labeled with Cy3 or Cy5 (CyDye, Agilent Technologies, USA) during the in vitro transcription process. 0.3 μg of Cy-labled cRNA was fragmented to an average size of about 50-100 nucleotides by incubation with fragmentation buffer at 60°C for 30 minutes.
|
| |
|
| Channel 2 |
| Source name |
BCPAP control
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Human Papillary thyroid cancer cell line, BCPAP
|
| Treatment protocol |
The cells were seeded (3 X 105 cells per well in 6-well plate) in antibiotic-free medium for 24 hours prior to transfection. DharmaFECT transfection reagent (Dharmacon RNA Technologies, Lafayette, CO) was used to transfect cells with mimic miR-146b (DharmaconRNATechnologies) for 24 hours at 50% confluence following the manufacturers instructions. The miRIDIAN miRNA mimic negative control (Dharmacon RNA Technologies) was used as control. Samples were collected after 24 hours of miRNA mimic transfections for quantification of miRNA. in antibiotic-free medium for 24 hours prior to transfection. DharmaFECT transfection reagent (Dharmacon RNA Technologies, Lafayette, CO) was used to transfect cells with mimicmiR- 146b (DharmaconRNATechnologies) for 24 hours at50% confluence following the manufacturer’s instructions. The miRIDIAN miRNA mimic negative control (Dharmacon RNA Technologies) was used as control. Samples were collected after 24 hours of miRNA mimic transfections for quantification of miRNA.
|
| Growth protocol |
The human PAPILLARY thyroid cancer cell line BCPAP was routinely cultured in RPMI 1640 containing 10% FBS, 100 U/mL penicillin, 100 g/mL streptomycin, and 2mML-glutamine(GIBCO,Rockville, MD) at 37°C in a humidified chamber containing 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from separated cell populations using Qiagen RNeasy Mini Kit (QIAGEN, Valencia, CA)
|
| Label |
Cy3
|
| Label protocol |
RNA from BCPAP control was labeled by Cy3 and RNA from BCPAP miR-146b-5p mimic transfected was labeled by Cy5. 0.2 μg of total RNA was amplified by a Low Input Quick-Amp Labeling kit (Agilent Technologies, USA) and labeled with Cy3 or Cy5 (CyDye, Agilent Technologies, USA) during the in vitro transcription process. 0.3 μg of Cy-labled cRNA was fragmented to an average size of about 50-100 nucleotides by incubation with fragmentation buffer at 60°C for 30 minutes.
|
| |
|
| |
| Hybridization protocol |
Correspondingly fragmented labeled cRNA is then pooled and hybridized to a Agilent SurePrint G3 Human GE 8×60K Microarray (Agilent Technologies, USA) at 65°C for 17 h. After washing and drying by nitrogen gun blowing, microarrays are scanned with an Agilent microarray scanner (Agilent Technologies, USA) at 535 nm for Cy3 and 625 nm for Cy5.
|
| Scan protocol |
Scanned images are analyzed by Feature extraction 10.5.1.1 software (Agilent Technologies, USA), an image analysis and normalization software is used to quantify signal and background intensity for each feature.
|
| Data processing |
Agilent Feature Extraction Software (v 8.5.1.1) was used for background subtraction and LOWESS normalization.
|
| |
|
| Submission date |
Dec 16, 2015 |
| Last update date |
Dec 17, 2015 |
| Contact name |
Chen Kai Chou |
| E-mail(s) |
vick0613@gmail.com
|
| Organization name |
Chang Gung Memorial Hospital-Kaohsiung Medical Center
|
| Street address |
No.123, Dapi Rd., Niaosong Dist
|
| City |
Kaohsiung |
| ZIP/Postal code |
83301 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE76050 |
Human Papillary thyroid cancer cell line, BCPAP control vs. miR-146b-5p mimic transfected |
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