 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 23, 2012 |
| Title |
MicroRNAs expression profiling during breast cancer progression |
| Organism |
Homo sapiens |
| Experiment type |
Non-coding RNA profiling by array
|
| Summary |
MicroRNAs (miRNAs) are known to be deregulated in human breast cancer (BC). The purpose of the current study was to investigate the expression of miRNAs in different stages of BC to assess their biological value in BC progression. MiRNA expression was assessed in a series of BC patients (n=7) with distinct stages of tumour progression (Normal, in-situ (DCIS), primary invasive BC and nodal metastases) to evaluate miRNA differential expression. We used an Agilent miRNA microarray based platform which uses miRBase 16 to screen for 1205 Homo sapiens (hsa) and 144 human viral miRNA candidates. To validate the microarray data, the expression of two deregulated miRNAs was measured by TaqMan quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR).
|
| |
|
| Overall design |
This study was conducted Formalin Fixed and Paraffin Embedded (FFPE) specimens from 7 female patients. Twenty μm thick FFPE sections were cut and mounted on PALM Membrane Slides. Under direct microscopic visualization, tissues of interest were micro-dissected using PALM non-contact Laser catapulsion instrument (P.A.L.M. Microlaser Technologies, Carl Zeiss Ltd). Total RNA, including miRNAs, was extracted with the TRIzol reagent (Invitrogen, supplied by Fisher Scientific UK Ltd) according to the manufacturer’s instructions. Total RNA was quantified with Quant-iT RiboGreen RNA Quantitation Kit (Fisher Scientific UK Ltd), which is an accurate method of measuring total RNA with a detection limit of 0.001-1ng/µl. RNA purity and RNA integrity number (RIN) were determined on an Agilent 2100 Bioanalyzer and RNA 6000 NanoLabChip Kits (both Agilent Technologies, Santa Clara, USA), and the RIN of all the samples was >2 but <3. This is expected for FFPE breast samples and is acceptable by Agilent Technologies, Santa Clara, USA, for FFPE samples to undergo miRNA microarray analysis. MiRNAs extracted from laser microdissected tissue components were profiled using Agilent microRNA microarray profiling system (Agilent Technologies, Santa Clara, USA). Total RNA samples were spiked using the MicroRNA Spike-In Kit (Agilent Technologies, Santa Clara, USA) to evaluate efficiencies of labelling and hybridisation. Total RNA was treated with Calf Intestinal Alkaline Phosphatase (CIP), and then 100 ng of total RNA was used per sample to initiate a labelling reaction. Ligation master mix for T4 RNA ligase, which includes Cyanine 3-Cytidine biphosphate (Cyanine 3-pCp) (Complete miRNA Labelling and Hyb Kit, Agilent Technologies, Santa Clara, USA) was used to label the dephosphorylated RNA. Cyanine-3-labelled miRNA samples were hybridised to human miRNA microarrays (Release 16.0, 8x60K) (Agilent Technologies, Santa Clara, USA) at 55°C for 20 hrs. Microarray slides were washed with increasing stringency (Gene Expression Wash Buffers, Agilent Technologies, Santa Clara), and subsequently dried with acetonitrile (Sigma-Aldrich, St. Louis, USA). Fluorescent signal intensities were detected on an Agilent Microarray Scanner (Agilent Technologies, Santa Clara, USA) using the Scan Control A.8.4.1 Software (Agilent Technologies, Santa Clara, USA) and extracted from the images with the Feature Extraction 10.7.3.1 Software (Agilent Technologies, Santa Clara, USA).
|
| |
|
| Contributor(s) |
Khoshnaw SM, Reis-filho JS, Lambros MB, Ball GR, Rakha EA, Abdel-Fatah TM, Nolan CC, Hodi Z, Macmillan RD, Ellis IO, Green AR |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
| |
| Submission date |
Jun 21, 2012 |
| Last update date |
Mar 08, 2013 |
| Contact name |
Sarkawt Khoshnaw |
| Organization name |
University of Nottingham
|
| Street address |
Queens Medical Centre
|
| City |
Nottingham |
| ZIP/Postal code |
NG7 2UH |
| Country |
United Kingdom |
| |
|
| Platforms (1) |
| GPL15019 |
Agilent-031181 Unrestricted_Human_miRNA_V16.0_Microarray 030840 (miRBase release 14.0 miRNA ID version) |
|
| Samples (28)
|
| GSM951044 |
Case one: normal breast tissue |
| GSM951045 |
Case one: ductal carcinoma in situ |
| GSM951046 |
Case one: invasive breast cancer tissue |
| GSM951047 |
Case one: metastatic breast cancer tissue |
| GSM951048 |
Case two: normal breast tissue |
| GSM951049 |
Case two: ductal carcinoma in situ |
| GSM951050 |
Case two: invasive breast cancer tissue |
| GSM951051 |
Case two: metastatic breast cancer tissue |
| GSM951052 |
Case three: normal breast tissue |
| GSM951053 |
Case three: ductal carcinoma in situ |
| GSM951054 |
Case three: invasive breast cancer tissue |
| GSM951055 |
Case three: metastatic breast cancer tissue |
| GSM951056 |
Case four: normal breast tissue |
| GSM951057 |
Case four: ductal carcinoma in situ |
| GSM951058 |
Case four: invasive breast cancer tissue |
| GSM951059 |
Case four: metastatic breast cancer tissue |
| GSM951060 |
Case five: normal breast tissue |
| GSM951061 |
Case five: ductal carcinoma in situ |
| GSM951062 |
Case five: invasive breast cancer tissue |
| GSM951063 |
Case five: metastatic breast cancer tissue |
| GSM951064 |
Case six: normal breast tissue |
| GSM951065 |
Case six: ductal carcinoma in situ |
| GSM951066 |
Case six: invasive breast cancer tissue |
| GSM951067 |
Case six: metastatic breast cancer tissue |
| GSM951068 |
Case seven: normal breast tissue |
| GSM951069 |
Case seven: ductal carcinoma in situ |
| GSM951070 |
Case seven: invasive breast cancer tissue |
| GSM951071 |
Case seven: metastatic breast cancer tissue |
|
| Relations |
| BioProject |
PRJNA169159 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE38867_RAW.tar |
235.6 Mb |
(http)(custom) |
TAR (of TXT) |
| GSE38867_raw.txt.gz |
64.2 Kb |
(ftp)(http) |
TXT |
| Processed data included within Sample table |
|
|
|
|
 |
Less...
More...