|
| Status |
Public on Nov 21, 2008 |
| Title |
Technical Analysis of Fundulus heteroclitus 384 cDNA microarray-.1X |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Fundulus heteroclitus cardiac tissue
|
| Organism |
Fundulus heteroclitus |
| Characteristics |
Tissue: F. heteroclitus cardiac tissue. Fish were acclimated to laboratory conditions for approximately 6 months.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from using 4.5M guanidinium thiocyanate, 2% N-lauroylsarcosine, 50mM EDTA, 25mM Tris-HCl, 0.1M beta-Mercaptoethanol and .2% Antifoam A. The extracted RNA was further purified using a Qiagen RNeasy Mini kit in accordance with the manufacturer’s protocols. The quantity and quality of the RNA was determined using a spectrophotometer (Nanodrop, ND-1000 V3.2.1) and a bioanalyzer (Agilent 2100). RNA was then converted into amino allyl labeled RNA (aRNA) using the Ambion Amino Allyl MessageAmp II aRNA Amplification kit. This method converts poly-A RNA into cDNA with a T7 RNA polymerase binding site and T7 is used to synthesize many new strands of RNA (in vitro transcription) (Eberwine, 1996).
|
| Label |
Cy3
|
| Label protocol |
During the in vitro transcription of aRNA amino allyl UTP (aaUTP) is incorporated into the elongating strand. aaUTP incorporation allows for the coupling of Cy3 (or Cy5) dyes (GE biosciences) onto aRNA for microarray hybridization. Dye labeled aRNA aliquots for each hybridization (30 pmol each of Cy3 and Cy5) were vacuum dried together and resuspended in 15ul hybridization buffer (final concentration of each labeled sample = 2 pmol/ul).
|
| |
|
| Channel 2 |
| Source name |
Fundulus heteroclitus cardiac tissue
|
| Organism |
Fundulus heteroclitus |
| Characteristics |
Tissue: F. heteroclitus cardiac tissue. Fish were acclimated to laboratory conditions for approximately 6 months.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from using 4.5M guanidinium thiocyanate, 2% N-lauroylsarcosine, 50mM EDTA, 25mM Tris-HCl, 0.1M beta-Mercaptoethanol and .2% Antifoam A. The extracted RNA was further purified using a Qiagen RNeasy Mini kit in accordance with the manufacturer’s protocols. The quantity and quality of the RNA was determined using a spectrophotometer (Nanodrop, ND-1000 V3.2.1) and a bioanalyzer (Agilent 2100). RNA was then converted into amino allyl labeled RNA (aRNA) using the Ambion Amino Allyl MessageAmp II aRNA Amplification kit. This method converts poly-A RNA into cDNA with a T7 RNA polymerase binding site and T7 is used to synthesize many new strands of RNA (in vitro transcription) (Eberwine, 1996).
|
| Label |
Cy5
|
| Label protocol |
During the in vitro transcription of aRNA amino allyl UTP (aaUTP) is incorporated into the elongating strand. aaUTP incorporation allows for the coupling of Cy3 (or Cy5) dyes (GE biosciences) onto aRNA for microarray hybridization. Dye labeled aRNA aliquots for each hybridization (30 pmol each of Cy3 and Cy5) were vacuum dried together and resuspended in 15ul hybridization buffer (final concentration of each labeled sample = 2 pmol/ul).
|
| |
|
| |
| Hybridization protocol |
Hybridization buffer consisted of 5X SSPE, 1% SDS, 50% formamide, 1mg/ml polyA, 1mg/ml sheared herring sperm carrier DNA, and 1mg/ml BSA. Slides were washed in sodium borohydride solution in order to reduce autofluorescence. Following rinsing, slides were boiled for 2 minutes and spin-dried in a centrifuge at 800 rpm for 3 minutes. Samples (15ul) were heated to 90ºC for 2 minutes, quick cooled to 42ºC, applied to the slide (hybridization zone area was 350mm2), and covered with a cover slip. Slides were placed in an airtight chamber humidified with paper soaked in 5X SSPE and incubated 24-48 hours at 42ºC.
|
| Scan protocol |
The microarray slides were scanned using ScanArray Express. The raw TIFF-image data was quantified using Imagene (v5). Genes with a fluorescence signal greater than 60,000 or less than the Ctenophore negative controls were eliminated from the analysis.
|
| Description |
Technical Analysis of Fundulus heteroclitus 384 cDNA microarray-.1X
|
| Data processing |
To adjust for systematic variation, gene expression values were first sum normalized and then loess normalized using Microarray Data Analysis System Software (MIDAS). For every gene, eight fluorescence values were captured; four for Cy3 and four for Cy5.
|
| |
|
| Submission date |
Sep 19, 2008 |
| Last update date |
Nov 20, 2008 |
| Contact name |
Cinda P Scott |
| E-mail(s) |
[email protected]
|
| URL |
http://crawford.rsmas.miami.edu/
|
| Organization name |
University of Miami
|
| Department |
Marine Biology and Fisheries
|
| Lab |
Crawford
|
| Street address |
4600 Rickenbacker Cswy
|
| City |
Miami |
| State/province |
FL |
| ZIP/Postal code |
33149 |
| Country |
USA |
| |
|
| Platform ID |
GPL7335 |
| Series (2) |
| GSE12858 |
Technical Analysis of Fundulus heteroclitus cDNA Microarrays, experiment A |
| GSE13687 |
Technical Analysis of Fundulus heteroclitus cDNA Microarrays |
|
