|
| Status |
Public on Mar 04, 2019 |
| Title |
pabociclib resistant cell RNA repeat 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
T47D cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Immortalized cell line
|
| Treatment protocol |
"M" samples were parental T47D cells, which had been cultured in conditioned medium for 48 hours.
|
| Growth protocol |
Parental T47D cells are were grown as directed by ATCC, resistant cells were cultured in the same way with the addition of 100 nM palbociclib to the medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using invitrogens "recoverall" kit according to the manufacturer guidelines
|
| Label |
Hy3
|
| Label protocol |
750 ng total RNA from both sample and reference was labeled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY LNA™ microRNA Hi-Power Labeling Kit, Hy3™/Hy5™ (Exiqon, Denmark) following the procedure described by the manufacturer.
|
| |
|
| Channel 2 |
| Source name |
reference
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: reference
|
| Treatment protocol |
"M" samples were parental T47D cells, which had been cultured in conditioned medium for 48 hours.
|
| Growth protocol |
Parental T47D cells are were grown as directed by ATCC, resistant cells were cultured in the same way with the addition of 100 nM palbociclib to the medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using invitrogens "recoverall" kit according to the manufacturer guidelines
|
| Label |
Hy5
|
| Label protocol |
750 ng total RNA from both sample and reference was labeled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY LNA™ microRNA Hi-Power Labeling Kit, Hy3™/Hy5™ (Exiqon, Denmark) following the procedure described by the manufacturer.
|
| |
|
| |
| Hybridization protocol |
The Hy3™-labeled samples and a Hy5™-labeled reference RNA sample were mixed pair-wise and hybridized to the miRCURY LNA™ microRNA Array 7th Gen (Exiqon, Denmark), which contains capture probes targeting all microRNAs for human, mouse or rat registered in the miRBASE 18.0. The hybridization was performed according to the miRCURY LNA™ microRNA Array Instruction manual using a Tecan HS4800™ hybridization station (Tecan, Austria). Spike-in controls were added in various concentrations in both the Hy3™ and the Hy5™ labeling reactions giving the opportunity to evaluate the labeling reaction, hybridization, and the performance of the array experiment in general. The high correlation (R2>0.95) of the signal intensities across all slides shown in the tables below for both the Hy3™ and Hy5™ channels indicates that both labeling and hybridization were successful. 52 different spike-in controls were added in concentrations covering the full signal range. Each spike-in control has 4 replicates of capture probes on the array.
|
| Scan protocol |
After hybridization the microarray slides were scanned and stored in an ozone free environment (ozone level below 2.0 ppb) in order to prevent potential bleaching of the fluorescent dyes. The miRCURY LNA™ microRNA Array slides were scanned using the Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA) and the image analysis was carried out using the ImaGene® 9 (miRCURY LNA™ microRNA Array Analysis Software, Exiqon, Denmark).
|
| Description |
All experiments were conducted at Exiqon Services, Denmark. The quality of the total RNA was verified by an Agilent 2100 Bioanalyzer profile. 750 ng total RNA from both sample and reference was labeled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY LNA™ microRNA Hi-Power Labeling Kit, Hy3™/Hy5™ (Exiqon, Denmark) following the procedure described by the manufacturer. The Hy3™-labeled samples and a Hy5™-labeled reference RNA sample were mixed pair-wise and hybridized to the miRCURY LNA™ microRNA Array 7th Gen (Exiqon, Denmark), which contains capture probes targeting all microRNAs for human, mouse or rat registered in the miRBASE 18.0. The hybridization was performed according to the miRCURY LNA™ microRNA Array Instruction manual using a Tecan HS4800™ hybridization station (Tecan, Austria). After hybridization the microarray slides were scanned and stored in an ozone free environment (ozone level below 2.0 ppb) in order to prevent potential bleaching of the fluorescent dyes. The miRCURY LNA™ microRNA Array slides were scanned using the Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA) and the image analysis was carried out using the ImaGene® 9 (miRCURY LNA™ microRNA Array Analysis Software, Exiqon, Denmark). The quantified signals were background corrected (Normexp with offset value 10, see Ritchie et al. 2007) and normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm.
0_Exiiqon files are the reference files
|
| Data processing |
The quantified signals were background corrected (Normexp with offset value 10, see Ritchie et al. 2007) and normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm. Data normalization was performed to minimize differences between the colors in an intensity dependent manner. Normalization was performed using Lowess (Locally Weighted Scatterplot Smoothing) normalization. Lowess uses a locally established regression to smooth the M/A (log ratio/log mean-intensity) scatterplot toward a linear distribution. The Lowess algorithm works under the assumption that the majority of the signals between samples do not differ and it enforces equal overall means on all signal intensities. Therefore, Lowess allows the correction of systematic deviations in the MA plot resulting in an intensity dependent adjustment of MA-data to a straight line. We have found this normalization to produce the best within-slide normalization and thereby minimize the intensity-dependent differences between the dyes. Array slide quality was also assessed using 52 different spole-in controls.
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| |
|
| Submission date |
Jul 26, 2018 |
| Last update date |
Mar 04, 2019 |
| Contact name |
Liam Cornell |
| E-mail(s) |
liam_cornell@dfci.harvard.edu
|
| Organization name |
Dana-Farber Cancer Institute
|
| Street address |
450 brookline avenue
|
| City |
boston |
| State/province |
MASSACHUSETTS |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL19128 |
| Series (2) |
| GSE117745 |
MicroRNA-mediated suppression of the TGF-ß pathway confers transmissible and reversible CDK4/6 inhibitor resistance (miRNA array) |
| GSE117748 |
MicroRNA-mediated suppression of the TGF-β pathway confers transmissible and reversible CDK4/6 inhibitor resistance |
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