Complementary RNA amplification methods enhance microarray identification of transcripts expressed in the C. elegans nervous system
© Watson et al; licensee BioMed Central Ltd. 2008
Received: 22 September 2007
Accepted: 19 February 2008
Published: 19 February 2008
DNA microarrays provide a powerful method for global analysis of gene expression. The application of this technology to specific cell types and tissues, however, is typically limited by small amounts of available mRNA, thereby necessitating amplification. Here we compare microarray results obtained with two different methods of RNA amplification to profile gene expression in the C. elegans larval nervous system.
We used the mRNA-tagging strategy to isolate transcripts specifically from C. elegans larval neurons. The WT-Ovation Pico System (WT-Pico) was used to amplify 2 ng of pan-neural RNA to produce labeled cDNA for microarray analysis. These WT-Pico-derived data were compared to microarray results obtained with a labeled aRNA target generated by two rounds of In Vitro Transcription (IVT) of 25 ng of pan-neural RNA. WT-Pico results in a higher fraction of present calls than IVT, a finding consistent with the proposal that DNA-DNA hybridization results in lower mismatch signals than the RNA-DNA heteroduplexes produced by IVT amplification. Microarray data sets from these samples were compared to a reference profile of all larval cells to identify transcripts with elevated expression in neurons. These results were validated by the high proportion of known neuron-expressed genes detected in these profiles and by promoter-GFP constructs for previously uncharacterized genes in these data sets. Together, the IVT and WT-Pico methods identified 2,173 unique neuron-enriched transcripts. Only about half of these transcripts (1,044), however, are detected as enriched by both IVT and WT-Pico amplification.
We show that two different methods of RNA amplification, IVT and WT-Pico, produce valid microarray profiles of gene expression in the C. elegans larval nervous system with a low rate of false positives. However, our results also show that each method of RNA amplification detects a unique subset of bona fide neural-enriched transcripts and thus a wider array of authentic neural genes are identified by the combination of these data sets than by the microarray profiles obtained with either method of RNA amplification alone. With its relative ease of implementation and greater sensitivity, WT-Pico is the preferred method of amplification for cases in which sample RNA is limiting.
Results and discussion
A comparison of two amplification methods, WT-Pico and IVT
Hybridization and amplification summaries for WT-Pico and IVT amplifications
Amplification method and sample type
Starting Material (ng)
Average Yield (ug)
No. of Chips
Average Intensity Values
Affymetrix average present calls/chip
Total number of transcripts identified by RNA amplification
Amplification method and sample type
Total number of present probesets
Total number of present genes
Neuron-enriched transcripts are identified in both the WT-Pico and IVT-amplified samples
To test the ability of the WT-Pico-amplified sample to detect differentially expressed transcripts, the pan-neural data set was compared to the reference profile obtained from all cells (see Methods). As expected, scatter plots reveal significant differences between these data sets with 1,625 transcripts showing elevated intensity values in the pan-neural sample vs 1,325 depleted mRNAs (Fig. 2e) [See Additional file 2]. (Similar results (Fig. 2f) were obtained by the IVT amplification method ). As an independent test of the validity of these data, the list of 1,625 transcripts showing elevated intensity values in the WT-Pico derived Pan-neural data set (i.e., "enriched genes") was compared to WormBase to identify the subset of transcripts previously described as expressed in neurons .
WT-Pico and IVT amplified targets reveal distinct neural transcripts
Expression of promoter-GFP reporters for transcripts enriched in larval pan-neural or A-class motor neuron data sets.
KOG (other description)
IVT Fold Change
WT-Pico Fold Change
Nuclear Hormone Receptor
Reticulocalbin, calumenin, DNA supercoiling factor, and related Ca2+-binding proteins of the CREC family (EF-Hand protein superfamily)
Predicted membrane protein
Uncharacterized conserved protein
ATP-dependent DNA ligase IV
GTPase Rab1/YPT1, small G protein superfamily, and related GTP-binding proteins
General control of a.a. synthesis 5-like 1
(putative nucleosome assembly factor)
The WT-Pico and IVT amplified samples identify C. elegans genes with homologs expressed in the mammalian brain
Microarray analysis of the IVT-amplified pan-neural sample detected 1,592 transcripts with elevated expression in C. elegans neurons  [See Additional file 3]. The independent microarray profile of these samples generated with the WT-Pico method has now identified an additional set of 581 neuron-enriched genes to yield a total of 2,173 transcripts that are highly expressed in the C. elegans nervous system (Fig. 7). Thus, the use of two alternative methods of RNA amplification has significantly expanded (~36%) the list of transcripts that are differentially expressed in C. elegans neurons. To assess the potential value of these additional data for studies of gene function in the nervous system, we identified a subset of genes in the WT-Pico-only list that are evolutionarily conserved but for which biochemical functions have not been previously assigned. This analysis yielded a total of 39 uncharacterized, highly conserved genes [See Additional file 7]. To determine if these transcripts are also expressed in mammalian neurons, we searched the Allen Brain Atlas, an online in situ hybridization database, for evidence of expression in the mouse brain . in situ data are available for 27 apparent homologs of the C. elegans genes on our list of WT-Pico-only enriched transcripts; 74% of these genes (20/27) show expression in the mouse brain. In the case of the IVT-only enriched transcripts, all seven of the uncharacterized, conserved genes for which in situ data are available in the Allen Brain Atlas are annotated as expressed in the mouse brain . These results support the idea that genes that are uniquely detected by one of these amplification methods are likely to encode authentic neural transcripts and that these combined data can provide potentially valuable clues to gene expression in the human brain.
3' bias does not account for differentially enriched targets identified by either WT-Pico or IVT
WT-Pico uses a combination of Poly-dT and random priming to amplify RNA. In contrast, the first round of the IVT is limited to Poly-dT priming. We speculated that this inherent difference in the amplification procedures might bias IVT towards probesets near the 3' end of a transcript. To test this hypothesis, each probeset identified as enriched by only IVT or only WT-Pico was mapped with the BLAT tool  to a unique chromosomal location in the WS170 assembly. From this position, we calculated the distance from the 3' end of the probeset to the 3' end of the gene it targets [See Additional file 8]. No statistically significant difference was found between the locations of the probesets unique to the WT-Pico method and those unique to the IVT method (p = 0.75). We therefore conclude that differential hybridization of WT-Pico vs IVT-generated targets is not due to a systematic bias of either amplified sample for probe sets near the 3' end of targeted transcripts. It should be noted however, that the probe sets in the GeneChip expression arrays used in this study are largely directed towards the 3'-end of the transcripts and therefore would not detect WT-Pico derived targets originating from more 5' regions. In the future, it will be interesting to examine transcripts that are independently detected with either IVT or WT-Pico-derived samples for potential nucleotide sequences that could exert differential effects on either RNA amplification or target hybridization.
We have confirmed that the WT-Pico method affords rapid and efficient RNA amplification with a higher fraction of present calls after microarray hybridization than targets amplified by the IVT protocol. The WT-Pico method is also technically easier to implement than IVT and requires significantly less time to perform. Although both approaches generate robust microarray profiles of gene expression in the C. elegans nervous system, a significant fraction of authentic neuron-enriched transcripts are uniquely identified by each of these methods of RNA amplification. Thus, the combined result obtained with both amplification strategies provides a more complete picture of neural gene expression than either sample alone. For cases in which RNA is limiting, as in the effort to profile single neuron types from C. elegans, the enhanced sensitivity of the WT-Pico method is advantageous.
Generating transgenic lines expressing GFP reporter genes
Promoter-GFP fusion genes were obtained from the Promoterome project and transgenic lines generated by microparticle bombardment as described . Additional file 9 contains a list of strains described in this paper.
mRNA-tagging and RNA amplification
The "in vitro transcribed" or "IVT" microarray data sets used in this paper are described in a previous publication. To generate these data sets, 25 ng of RNA from three pan-neural replicates and from five independent N2 (reference) samples was amplified by the IVT method . 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 User Guides . Two of the previously prepared reference RNA preparations did not amplify by WT-Pico. Two additional samples were isolated by the mRNA-tagging method from the pan-neural transgenic line, SD1241 for WT-Pico amplification to yield a total of five pan-neural replicates and three reference samples for the "WT-Pico" profiles. Thus, six of the eight pan-neural and reference data sets generated by each of the RNA amplification methods (IVT or WT-Pico) were obtained from identical RNA samples. A quantitative comparison of microarray results obtained from the two new pan-neural RNA samples (DMM10 and DMM11) used for the WT-Pico amplification vs the originally isolated pan-neural preparations (DMM2, DMM3, DMM4) also used for the IVT amplification  showed a broadly similar distribution of intensity values (R2 > 0.91) (see Fig 2).
Microarray data analysis
Microarray data were processed as described [8, 9]. 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) . 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) . 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) (, this work). The data discussed in this manuscript are available in the NCBI Gene Expression Omnibus, series accession number GSE9485.
Annotation of data sets and additional data analysis
Annotation was performed as previously described using WormBase Release 170 http://WS170.wormbase.org. Affymetrix GeneChip Operating Software (GCOS) was used to calculate the average number of present calls for each probe set (Table 1) . Present calls listed in Table 2 and used to calculate Fig. 5 were identified with a Perl script (consensus.pl) [See Additional file 10]. For a given sample (e.g. IVT pan-neural) a transcript was scored "present" if called present in all replicates. For Fig. 5., 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.
RMA normalized intensity values for all data sets were imported into GeneSpring GX 7.3  to generate the line graphs shown in Fig. 6. Each Experimental data set 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 . 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.
Microscopy and identification of GFP expressing cells
GFP-expressing animals were visualized by Differential Interference Contrast (DIC) and epifluorescence optics in either a Zeiss Axioplan or Axiovert compound microscope. Digital images were recorded with CCD cameras (ORCA I or ORCA ER, Hamamatsu Corporation, Bridgewater, NJ).
We thank Christian Schaffer and Marilyn Ritchie for updating Perl scripts used to annotate the data; Braden Boone and John Mote of the Vanderbilt Microarray Shared Resource (VMSR) for help with microarray experiments and with GeneSpring software, Denis Dupuy and Marc Vidal for Promoterome constructs, Kylee Spencer for advice on statistical methods, and Damian Marlee for help with database management. This work was supported by NIH grants R01 NS26115, P01 DK58212 and HG004263 (DMM); F31 NS049743 and T32 MH64913 (JDW), P30 CA68485, P60 DK20593, P30 DK58404, HD15052, P30 EY08126, and P01 HL6744.
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