|
| Status |
Public on Aug 31, 2015 |
| Title |
CLL Cellular RNA 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
RNA extracted from CLL cells patient 2
|
| Organism |
Homo sapiens |
| Characteristics |
rna source: Cellular RNA from CLL patient 2
cell type/component: isolated B cells from fresh blood CLL2
tissue: blood
|
| Growth protocol |
CLL cells were cultured for 48h and media supernatant was harvested. Exosomes were purified from this supernatant.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extractions were performed using the miRNeasy kit (Qiagen). Briefly, the quality of the total RNA was verified by an Agilent 2100 Bioanalyzer.
|
| Label |
Hy3
|
| Label protocol |
We used dual colour hybridizations and we used the common reference approach which each sample whether exosomal RNA or cellular RNA was compared to common reference sample (a pool of 23 CLL cells).
|
| |
|
| Channel 2 |
| Source name |
Pooled reference sample
|
| Organism |
Homo sapiens |
| Characteristics |
reference type: a collection of 23 RNA extracted from 23 CLL cell samples
|
| Growth protocol |
CLL cells were cultured for 48h and media supernatant was harvested. Exosomes were purified from this supernatant.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extractions were performed using the miRNeasy kit (Qiagen). Briefly, the quality of the total RNA was verified by an Agilent 2100 Bioanalyzer.
|
| Label |
Hy5
|
| Label protocol |
We used dual colour hybridizations and we used the common reference approach which each sample whether exosomal RNA or cellular RNA was compared to common reference sample (a pool of 23 CLL cells).
|
| |
|
| |
| Hybridization protocol |
The Hy3-labelled exosome samples or CLL cell samples and a Hy5-labelled CLL cell samples pool (n=23; common reference sample) were hybridized to the miRCURY LNA array (Exiqon miRCURYTM LNA array v.5). The hybridization and all steps of the image analysis and normalisation of data were carried out by Exiqon.
|
| Scan protocol |
The hybridization and all steps of the image analysis and normalisation of data were carried out by Exiqon.
|
| Description |
a pool of cellular RNA from 23 CLL individuals
|
| Data processing |
Median data for Hy3 and Hy5 was calculated from the replicated measurements on the same slide (SD-values were calculated from 4 replicate spots).
Different normalization methods such as lowess and quantile were performed to see the best method to use to compare the results between the exosomal and the cellular samples. We were unable to find a suitable normalizing method for both exosomes and cells thus it was found that the best method was to use the raw intensity data from the arrays after background correction. Other method skewed the data set and provided unreliable results
The supplementary file "LME_background-subtracted_Hy3.txt" contains LogMedianExpression (LME) of Hy3 background-subtracted raw values.
|
| |
|
| Submission date |
Jul 16, 2015 |
| Last update date |
Aug 31, 2015 |
| Contact name |
Mosavar Farahani |
| E-mail(s) |
mosavar@liv.ac.uk
|
| Phone |
+44 151 7064144
|
| Organization name |
University of Liverpool
|
| Department |
Molecular and Clinical Cancer Medicine
|
| Lab |
Haematology
|
| Street address |
Daulby Street
|
| City |
Liverpool |
| ZIP/Postal code |
L69 3GA |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL20657 |
| Series (2) |
| GSE70994 |
CLL exosomes modulate the transcriptome and behaviour of recipient stromal cells and are selectively enriched in miR-202-3p [mirBase 13] |
| GSE70996 |
CLL exosomes modulate the transcriptome and behaviour of recipient stromal cells and are selectively enriched in miR-202-3p |
|
