|
| Status |
Public on Nov 01, 2007 |
| Title |
Larval Reference, biological rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
WT-Pico amplified RNA obtained from all cells by performing a control IP
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
Wildtype (N2 isolate) nematodes
|
| Treatment protocol |
For mRNA-tagging samples, synchronized L2 larvae were fixed in 0.5% formaldehyde, homogenized by passing through a French press (6000 psi on minicell) and using a Dounce homogenizer, and the cell-free extract isolated after a low-speed and high-speed spin. The FLAG-PAB bound RNA was isolated by immunoprecipitating with anti-FLAG antibodies, reversing the crosslink in a Tris buffer at 65 degrees Celcius, and TriZOL extracted.
|
| Growth protocol |
A synchronized population of larvae was obtained by the following method. Gravid adults expressing either F25B3.3::FLAG::PAB-1 (pan-neural) or unc-4::FLAG::PAB-1 (A-class) transgenes were subjected to bleach/NaOH treatment to release embryos. Embryos were harvested and allowed to hatch o/n in M9 liquid. Hatched L1 larvae were harvested and placed on plates containing food to restart development. After 22-24hr, when >80% of larvae had reached the mid-L2 stage, larvae were harvested for RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For mRNA-tagging samples, following elution/crosslink, RNAs were isolated by TriZOL extraction and isopropanol precipitation (following manufacturer's protocol).
|
| Label |
biotin
|
| Label protocol |
A 2-round IVT protocol (modified from the Affymetrix small-sample protocol) was used to convert 25ng of RNA into biotinylated aRNA (described in Von Stetina, Watson, et al 2007). For WT-Pico, 2 ng of these RNAs was amplified using version 1 of the WT-Ovation Pico System, which combines WT-Ovation™ Pico RNA Amplification System and target preparation according to fragmentation and labeling section of Ovation™ Biotin RNA Amplification and Labeling System as described in the respective User Guides (http://www.nugeninc.com/html/04_technical_resources1.html)
|
| |
|
| Hybridization protocol |
affymetrix
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 7G.
|
| Description |
Gene expression data from all wildtype mid-L2 nematode cells
|
| Data processing |
Microarray data were processed as described [8, 33]. Briefly, intensity values from each hybridization were scaled vs a global average signal from the same array and normalized by Robust Multichip Average analysis (RMA) [34]. To identify differentially expressed transcripts, normalized intensity values from the Pan-neural data sets were compared to a Reference (from all larval cells) using Significance Analysis of Microarray software (SAM) [35]. A two-class unpaired analysis of the data was performed to identify neuron-enriched genes. Pan-neural enriched transcripts in the IVT and WT-Pico-derived data set were defined as 1.5X elevated vs the Reference at a False Discovery Rate (FDR) = 3%. An earlier report describing the IVT-amplified Pan-neural data set utilized a more stringent FDR of 1% and therefore identified a smaller number of Pan-neural enriched transcripts (1,562 vs 1,592 in this study) (Von Stetina et al. Watson et al. 2007).
Annotation was performed as previously described using WormBase Release 170 (WS170.wormbase.org). Affymetrix GeneChip Operating Software (GCOS) was used to calculate the average number of present calls in for each probe set (Table 1) [25]. Present calls listed in Table 2 and used to calculate Fig. 6 were identified with a perl script (consensus.pl) (Additional Data File 10). For a given sample (e.g. IVT Pan-neural) a transcript was scored “present” if it was called present in all replicates. For Fig 6., RMA-normalized intensities for these present genes were averaged across all replicates. Average Pan-neural/Reference intensities were calculated for WT-Pico and IVT data and log2 transformed. The coefficient of determination (R2) for the resulting scatter plot was calculated in Microsoft Excel. Mismatch (MM) intensities were compared against Perfect Match (PM) intensities for both Pan-neural and reference and samples using the Bioconductor [36] Affy package [36] for Fig 3 . RMA normalized intensity values for all datasets were imported into GeneSpring GX 7.3 (http://www.chem.agilent.com/scripts/pds.asp?lpage=27881) to generate the line graphs shown in Fig 7. Each Experimental dataset was normalized vs the average intensity value for each probe set in the corresponding Reference data set and plotted as log (Experimental/Reference). Each vertical line represents an individual replicate for each Experimental sample. p-values for total yield, number of present genes, and perfect vs mismatch probes were calculated using a two-tailed t-test with unequal variance. 3’ bias analysis IVT-only and WT-Pico only enriched transcripts were examined for 3’ bias. C_elegans_target.fa was downloaded from Affymetrix. This file contains the reference sequence for each probeset on the array. The file Caenorhabditis_elegans.WB170.45.dna.seqlevel.fa was downloaded from Ensembl (Ensembl 45, based on Wormbase 170). Probesets were aligned to chromosomes using BLAT [31]. Where multiple alignments were found, the alignment that covered the longest portion of the probeset sequence was chosen. The genes and chromosomal locations of those genes were downloaded using Ensembl 45. The probeset distance from the 3’ end of the gene was calculated. For genes on the (+) strand, the distance is given as (Gene End) – (Probeset End). For genes on the (-) strand, the distance is given as (Probeset Start) – (Gene Start). For probesets that correspond to multiple genes, the gene with the smallest absolute value of 3’ distance was chosen. p value was calculated using a 2-tailed t-test with equal variance.
|
| |
|
| Submission date |
Oct 31, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
David Miller |
| E-mail(s) |
david.miller@vanderbilt.edu
|
| Phone |
6153433447
|
| Fax |
6159365673
|
| URL |
http://exploration.vanderbilt.edu/news/news_worm.htm
|
| Organization name |
Vanderbilt University
|
| Department |
Cell and Developmental Biology
|
| Street address |
465 21st Avenue South
|
| City |
Nashville |
| State/province |
TN |
| ZIP/Postal code |
37232-8240 |
| Country |
USA |
| |
|
| Platform ID |
GPL200 |
| Series (1) |
| GSE9485 |
cRNA amplification methods enhance microarray identification of transcripts expressed in the nervous system |
|
