|
| Status |
Public on Jul 21, 2006 |
| Title |
slide46_M |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source Name |
Anthers dissected from the upper floret of spikelets from maize tassels and staged at the early microspore developmental stage by light microscopy
|
| Organism(s) |
Zea mays |
| Characteristics |
Inbred line: Ky21;Floret: Upper;Developmental stage: Early microspore;Sample name: EM2u
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol reagent (Invitrogen, Carlsbad, CA) as per manufacturer's instructions
|
| Label |
cy3
|
| Label protocol |
Fluorescent targets were synthesized and hybridized as described at http://schnablelab.plantgenomics.iastate.edu/resources/protocols/. Only targets that contained more than 3000 picomoles of cDNA, more than 60 picomoles of Cy dye and more than one dye molecule per 50 bases were used for hybridizations.
|
| |
|
| Channel 2 |
| Source Name |
Anthers dissected from the lower floret of spikelets from maize tassels and staged at the early microspore developmental stage by light microscopy
|
| Organism(s) |
Zea mays |
| Characteristics |
Inbred line: Ky21;Floret: Lower;Developmental stage: Early microspore;Sample name: EM2L
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol reagent (Invitrogen, Carlsbad, CA) as per manufacturer's instructions
|
| Label |
cy5
|
| Label protocol |
Fluorescent targets were synthesized and labeled as described in the "cDNA microarray protocol" file at http://schnablelab.plantgenomics.iastate.edu/resources/protocols/. Only targets that contained more than 3000 picomoles of cDNA, more than 60 picomoles of Cy dye and more than one dye molecule per 50 bases were used for hybridizations.
|
| |
|
| |
| Hybridization protocol |
Fluorescent targets were hybridized as described in the "cDNA microarray protocol" file at http://schnablelab.plantgenomics.iastate.edu/resources/protocols/.
|
| Scan protocol |
Each microarray chip (48 total) was scanned six times in ascending amounts of laser power and PMT gain with a ScanArray 5000 (Packard, Meriden, CT). Three scans from each chip were selected based on the median value of the natural log of the signal median for all spots on the slide. The values for the low, medium, and high scan intensity data sets were 5.7, 7.2 and 8.7, respectively.
|
| Description |
RNA was isolated from eight to twenty anthers per each individual tassel using Trizol reagent (Invitrogen, Carlbad, CA) as per manufacturer's instructions. Equal amounts of RNA from one to four individuals per stage per floret type (i.e., upper or lower) were pooled randomly to generate one biological replicate. In total, 24 biological replicates (two biological replicates per stage per floret type) were generated. Approximately 100 ng of total RNA from each biological replicate were used as starting material for T7-based linear RNA amplification, performed as described by Nakazono et al. (2003). Each biological replicate yielded between 30 and 50 micrograms of amplified RNA (aRNA). Approximately 3 micrograms of aRNA for each sample was indirectly labeled with Cy dye and hybridized to the GPL3021 sample platform.
|
| Data processing |
An R implementation of the lowess normalization method (Dudoit and Fridlyand, 2002) was used to normalize the two channels for each combination of slide and scan intensity. The lowess normalized data from each scan was used to conduct a mixed linear model analysis separately for each of 12,160 spots using a strategy similar to that of Wolfinger et al., (2001)
|
| |
|
| Submission date |
Sep 22, 2005 |
| Contact name |
Patrick S. Schnable |
| E-mail(s) |
schnable@iastate.edu
|
| Phone |
515-294-0975
|
| Organization name |
Iowa State University
|
| Street address |
2035B Roy J Carver Co-Lab
|
| City |
Ames |
| State/province |
IA |
| ZIP/Postal code |
50011 |
| Country |
USA |
| |
|
| Platform ID |
GPL3021 |
| Series (1) |
| GSE3017 |
Global gene expression profiling of developing maize anthers |
|
| Data table header descriptions |
| ID_REF |
|
| VALUE |
-[INV_VALUE] |
| Ch1_Signal Mean |
Pixel intensity avaeraged over the local signal region for green channel (Cy3) |
| Ch1_Background Mean |
Pixel intensity avaeraged over the local background region for green channel (Cy3) |
| Ch1_Signal Median |
Median pixel intensity computed over the local signal region for green channel (Cy3) |
| Ch1_Background Median |
Median pixel intensity computed over the local background region for green channel (Cy3) |
| Ch2_Signal Mean |
Pixel intensity avaeraged over the local signal region for red channel (Cy5) |
| Ch2_Background Mean |
Pixel intensity avaeraged over the local background region for red channel (Cy5) |
| Ch2_Signal Median |
Median pixel intensity computed over the local signal region for red channel (Cy5) |
| Ch2_Background Median |
Median pixel intensity computed over the local background region for red channel (Cy5) |
| Ch1_Norm |
Background corrected and normalized log value of green channel(Cy3) |
| Ch2_Norm |
Background corrected and normalized log value of red channel(Cy5) |
| INV_VALUE |
Normalized log ratio value of background corrected intensities of red channel and green channel |
