|
| Status |
Public on Oct 18, 2010 |
| Title |
Norm.2.col.Ref1.1 (NCode) |
| Sample type |
RNA |
| |
|
| Source name |
Total RNA with small fraction
|
| Organism |
Mus musculus |
| Characteristics |
sample: Reference 1
tissue: pool of equal amounts of total RNA from mouse testicle, ovary and 10-12 day embryo.
sample type: Reference RNA was developed from FirstChoice® mouse Total RNA including the small fraction (Catalogue # AM7800-AM7828, Ambion, Streetsville, ON)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA containing the small fraction was isolated from each sample using the miRVana miRNA Isolation Kit (Ambion), Streetsville, ON, Canada). Quality was confirmed using the Agilent 2100 Bioanalyzer and RNA Nano 6000 chips.
|
| Label |
Alexa Fluor 3 , Alexa Fluor 5
|
| Label protocol |
0.5 ug of total reference RNA was spiked with 40 fmoles NCode Microarray Positive Control (Invitrogen) and labelled using the NCode Rapid miRNA Labelling System according to manufacturer’s instruction (Invitrogen). Briefly, polyA polymerase is used to add a poly(A) tail to the RNA, to which is then ligated a DNA polymer carrying ~15 molecules of the fluorophore (Alexa Fluor® 3 or 5). Ligation is mediated by an oligo(dT) bridge whose sequence matches both the poly(A) tail of the RNA, and the fluorophore-labelled DNA polymer.
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| |
|
| Hybridization protocol |
Arrays were hybridized 10 hours at 52°C. In a modification of the standard protocol, hybridizations were performed using Agilent hybridization chambers within a rotisserie oven, which required dilution of the hybridization mix to a total volume of 120 ul, by addition of BSA (8 mg/ml) and Enhanced Hybridization Buffer (Invitrogen). Arrays were washed and dried by centrifugation according to manufacturer’s instructions. To protect dyes against ozone-mediated degradation, 0.1 mM dithiothreitol was added to each wash solution, and slides were dried and stored in sealed tubes containing 20 ul of ≥ 98% 2-mercaptoethanol, ensuring that there was no contact between the reducing agent and the array.
|
| Scan protocol |
Arrays were scanned with a GenePix 4000B laser scanner (Molecular Devices) with a scan resolution of 5 um, and PTM between 620 to 660 V. Signal intensity was determined using Imagene 8.0 image analysis software (Biodiscovery, Inc.).
|
| Description |
Alexa Fluor 3: 3779_reformat.txt
Alexa Fluor 5: 3863_reformat.txt
|
| Data processing |
Median signal intensities were lowess normalized for the 2 colour analysis and cyclic lowess normalized for the one colour analysis Alexa Fluor 5 channel only). Technical replicates for each miRNA level were averaged using the median. Data was flagged if the raw signal intensity was less than the mean plus three standard deviations of the negative controls or if the median of the technical replicates was flagged to be in the background.
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| |
|
| Submission date |
Dec 29, 2009 |
| Last update date |
Oct 18, 2010 |
| Contact name |
Andrew Williams |
| Organization name |
Health Canada
|
| Street address |
50 Columbine
|
| City |
Ottawa |
| State/province |
ON |
| ZIP/Postal code |
K1A OK9 |
| Country |
Canada |
| |
|
| Platform ID |
GPL9838 |
| Series (1) |
| GSE19669 |
Cross-platform correlation analysis of global microRNA expression technologies |
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