|
| Status |
Public on Nov 21, 2009 |
| Title |
KK2_2_normalized,dye-swap |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
KK2, biological replicate 2, female
|
| Organism |
Drosophila ananassae |
| Characteristics |
strain: KK2
gender: female
age: 4-5 days
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol reagent (Invitrogen) and the manufacturer's protocol and RNA was reverse transcribed to cDNA using 25 microgramm of total RNA and an anchored oligo(dT) primer.
|
| Label |
Alexa Fluor 647 (Cy5)
|
| Label protocol |
cDNA was labelled with fluorescent dyes (Alexa Fluor 555 and 647) using the amino-allyl labelling system (Invitrogen)
|
| |
|
| Channel 2 |
| Source name |
KK2, biological replicate 2, male
|
| Organism |
Drosophila ananassae |
| Characteristics |
gender: male
age: 4-5 days
strain: KK2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol reagent (Invitrogen) and the manufacturer's protocol and RNA was reverse transcribed to cDNA using 25 microgramm of total RNA and an anchored oligo(dT) primer.
|
| Label |
Alexa Fluor 555 (Cy3)
|
| Label protocol |
cDNA was labelled with fluorescent dyes (Alexa Fluor 555 and 647) using the amino-allyl labelling system (Invitrogen)
|
| |
|
| |
| Hybridization protocol |
Labelled male and female cDNA was competitively hybridized to the arrays for 20 hours at 42°C.
|
| Scan protocol |
Arrays were scanned using a Genetix aQuire 2-laser microarray scanner and the Genetix Qscan software.
|
| Description |
PCR-amplicon microarrays with 148 genes + gDNA controls were produced and hybridized
|
| Data processing |
Quality control
1. Only spots with mean intensity 20% above the mean local background intensity in at least one of the channels were used
This is a bit more lenient than requiring 1 SD above background (typically the SD is 20-30% of the mean)
2. For any one array, it was required that at least half of the replicate spots per gene (in this case 4) had good signal as defined by #1 above.
If so, the median Cy3/Cy5 ratio (after normalization) of all good replicate spots was used.
Normalization
1. The signal for each spot was calculated by subtracting the MEDIAN local background from the MEAN spot intensity.
If the above result was negative for one channel, that channel was given a value of 0.5 (MINIMUM approach of limma).
2. For each replicate array (162 spots, 1 per gene), the Cy3/Cy5 ratio was calculated for each of the gDNA control spots
3. The mean of the above Cy3/Cy5 ratios was taken and used as a "corrector".
This corrector was multiplied by the raw Cy5 value of each spot to get a corrected Cy5 value that was used to calculate the ratio.
Note: this is a local normalization, with a different corrector for each replicate array. There are 8 replicate arrays per hybridization.
|
| |
|
| Submission date |
Nov 19, 2009 |
| Last update date |
Nov 20, 2009 |
| Contact name |
Sonja Grath |
| E-mail(s) |
grath@bio.lmu.de
|
| Organization name |
LMU
|
| Department |
Department Biology II
|
| Lab |
Evolutionary and Functional Genomics
|
| Street address |
Grosshaderner Str. 2
|
| City |
Planegg-Martinsried |
| ZIP/Postal code |
82152 |
| Country |
Germany |
| |
|
| Platform ID |
GPL9699 |
| Series (1) |
| GSE19096 |
Molecular evolution of sex-biased genes in the Drosophila ananassae subgroup |
|
