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METH O D O L O G Y AR T I C L E Open Access
Multi-targeted priming for genome-wide gene
expression assays
Aleksandra B Adomas
1
, Francesc Lopez-Giraldez
1
, Travis A Clark
1
, Zheng Wang
1
, Jeffrey P Townsend
2*
Abstract
Background: Complementary approaches to assaying global gene expression are needed to assess gene
expression in regions that are poorly assayed by current methodologies. A key component of nearly all gene
expression assays is the reverse transcription of transcribed sequences that has traditionally been performed by
priming the poly-A tails on many of the transcribed genes in eukaryotes with oligo-dT, or by priming RNA
indiscriminately with random hexamers. We designed an algorithm to find common sequence motifs that were
present within most protein-coding genes of Saccharomyces cerevisiae and of Neurospora crassa, but that were not
present within their ribosomal RNA or transfer RNA genes. We then experimentally tested whether degenerately
priming these motifs with multi-targeted primers improved the accuracy and completeness of transcriptomic
assays.
Results: We discovered two multi-targeted primers that would prime a preponderance of genes in the genomes
of Saccharomyces cerevisiae and Neurospora crassa while avoiding priming ribosomal RNA or transfer RNA.
Examining the response of Saccharomyces cerevisiae to nitrogen deficiency and profiling Neurospora crassa early
sexual development, we demonstrated that using multi-targeted primers in reverse transcription led to superior
performance of microarray profiling and next-generation RNA tag sequencing. Priming with multi-targeted primers
in addition to oligo-dT resulted in higher sensitivity, a larger number of well-measured genes and greater power to
detect differences in gene expression.
Conclusions: Our results provide the most complete and detailed expression profiles of the yeast nitrogen
starvation response and N. crassa early sexual development to date. Furthermore, our multi-targeting priming
methodology for genome-wide gene expression assays provides selective targeting of multiple sequences and
counter-selection against undesirable sequences, facilitating a more complete and precise assay of the transcribed
sequences within the genome.
Background
Gene expression levels have been quantified by numer-
ous procedures, including reverse transcription (RT)-
PCR [1], sequencing of expressed sequence tags [2],
serial analysis of gene expression [3], microarray hybri-
dization [4], and massively parallel signature sequencing
[5]. Rapid development of platforms has improved
throughput, but also generated strong demand for
enhanced sensitivity and measurement accuracy. For
nearly all expression assays, reverse transcription from
messenger RNA (mRNA) to complementary DNA
(cDNA) is a key step of the process that contributes less
experimental variance than biological growth and har-
vest, but greater experimental variance than hybridiza-
tion [[6], but see also [7]]. Throughput of the reaction
may be biased by secondary and tertiary structures of
mRNA, affinities specific to the reverse transcriptase,
inhibitors present in the sample, priming strategy, and
variation in priming efficiency [8]. The most common
priming strategies utilize oligo-dT primers, random pri-
mers, or gene-specific primers. When oligo-dT primers
are used for reverse transcription, RNA secondary struc-
ture and variation in poly(A) tail length may result in
gene amplification 3′bias [9]. Random primers, typically
used in prokaryotic systems, fail to discriminate between
* Correspondence: Jeffrey.Townsend@Yale.edu
2
Department of Ecology and Evolutionary Biology, Program in
Computational Biology and Bioinformatics, and Microbiology Graduate
Program, Yale University, 165 Prospect St, New Haven, CT 06511, USA
Full list of author information is available at the end of the article
Adomas et al.BMC Genomics 2010, 11:477
http://www.biomedcentral.com/1471-2164/11/477
© 2010 Adomas et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution L icense (http://creati vecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and repro duction in
any medium, provided the original work is properly cited.
mRNA and the preponderance of RNA in the form of
ribosomal (rRNA) or transfer RNA (tRNA). Random
hexamers, the most commonly employed, amplify only
fraction of the transcriptome, comparing with random
pentadecamers [10]. However, random oligonucleotides
of any size also prime abundant rRNA and tRNA that
can lead to high background and misleading signal.
Ribosomal RNA (rRNA) sequences in many prokar-
yotes are GC rich relative to the genome at large and
are highly conserved. These properties have been used
to design non-random hexamers (HD/DHTTTT) to
prime reverse transcription reactions [11]. The result
was a counter-selective synthesis of cDNA correspond-
ing to mRNA from prokaryotic total RNA extractions.
In contrast, application of gene-specific primers on a
genomic scale requires synthesis of multiple primers. An
algorithm to predict the minimal number of non-degen-
erate genome-directed primers that specifically anneal to
all genes in a given genome has been designed and suc-
cessfully applied in bacteria [12].
Another recently developed method relies on a collec-
tion of short, computationally selected oligonucleotides
(’not-so-random’(NSR) primers) to obtain full-length,
strand-specific representation of nonribosomal RNA
transcripts [13]. Selective enrichment of non-rRNA tar-
gets was achieved by computationally subtracting rRNA
priming sequences from a random hexamer library. The
presence of rRNA and tRNA plagues most mRNA puri-
fication procedures due to their relative abundance,
leading to non-specific interactions like rRNA adsorp-
tion to the oligo-dT matrix, or hybridization of rRNA
and mRNA sequences [14].
Here we describe an alternate strategy, multi-targeted
priming (MTP), that allows for selective amplification of
chosen sequences. A degenerate oligonucleotide comple-
mentary to selected mRNAs and absent in both rRNA
or tRNA was identified allowing for selective transcrip-
tion of mRNA. To demonstrate the power of MTP, spe-
cies-specific primers were designed and tested on RNA
from Saccharomyces cerevisiae exposed to nitrogen defi-
ciency, and on RNA from Neurospora crassa during
early sexual development following nitrogen depletion.
When primary nitrogen sources are not available or are
present in concentrations low enough to limit growth,
many different nitrogen sources can be used. Utilization
of secondary nitrogen sources is highly regulated, and
nearly always requires the synthesis of a set of pathway-
specific catabolic enzymes and permeases [15]. Several
studies have shown the induction of a common suite of
effector genes during growth of fungal plant pathogens
under nitrogen-starved conditions in vitro and during
growth in planta [16,17]. As a consequence, nitrogen-
starved media has become a model for the environment
that a pathogen encounters during growth in planta
[16]. Furthermore, nitrogen uptake and exchange are
key processes for ectomycorrhizal interactions that are
established between the root systems of terrestrial plants
and hyphae from soil-borne fungi [18,19]. Finally, nitro-
gen deficiency has been associated with major problems
encountered in contemporary wine making [20], espe-
cially those related to slow and incomplete fermenta-
tions [21]. Therefore, the response of budding yeast
exposed to nitrogen starvation has been of interest in
light of nutrient depletion during wine fermentation.
Neurospora crassa is a heterothallic filamentous fun-
gus that undergoes a complex pattern of sexual differen-
tiation to form the female reproductive structure
(protoperithecium) when subjected to conditions of
nitrogen starvation, light, and low temperature [22]. A
large number of genes affecting sexual development
have been identified by mutation [23,24], but large-scale
transcript profiling has not been performed.
Here we show for these applications that the addition
of an MTP provides superior sensitivity and precision to
microarray transcript profiling compared to sole use of
oligo-dT primers. We corroborate these results for high
throughput RNA tag sequencing, incorporating modifi-
cation of the Illumina Digital Gene Expression protocols
to facilitate use of MTPs.
Results
Transcript profiling of S. cerevisiae grown in nitrogen-
poor conditions
We identified a 12-nucleotide (nt) degenerate sequence
that occurs one or more times in 76% of 6608 mRNAs
present in budding yeast, Saccharomyces cerevisiae,but
that is absent in rRNA or tRNA. The corresponding
degenerate primer was NDKTBBBBDWGS. Among the
5039 yeast ORFs containing the identified degenerate
sequence, 86% featured one to five exact priming sites
and the remaining sequences featured six to 44 priming
sites (see Additional File 1). On average, there were 3.2
priming sites per gene. Priming for reverse transcription
is not highly specific [8], thus MTPs may prime nearly
all genes with some frequency. Nonetheless, the MTP
and an equal proportion of oligo-dT primers were sup-
plied for the reverse transcription step during the micro-
array target labeling procedure, to achieve the greatest
coverage possible.
Contrasting yeast grown in nitrogen-poor and nitro-
gen-rich conditions, priming by MTP increased number
of well measured genes, recorded after data normaliza-
tion, by 31% over oligo-dT priming (Table 1; see Addi-
tional File 2). A logistic regression indicates that a
model featuring the number of MTP binding sites in a
gene is a statistically significantly better predictor of
whether a statistically significant difference in expression
will be observed when using MTPs than is a model
Adomas et al.BMC Genomics 2010, 11:477
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without a slope with regard to number of MTP binding
sites (P < 0.0001, slope 0.05). However, a model featur-
ing the number of MTP binding sites is also a statisti-
cally significant predictor of the probability of a
statistically significant call when oligo-dT alone is used
(P = 0.02, slope 0.03). More saliently, use of the MTP
increased the number of genes that were identified as
significantly differentially expressed by 66%.
We analyzed the frequency of MTP priming sites
among genes identified as significantly differentially
expressed in nitrogen-depleted yeast. Out of 1738 genes
indicated by MTPs exclusively as differentially
expressed, 342 did not have an MTP recognition site
and 1396 genes contained one to 29 priming sites.
Among 316 genes exclusively identified by oligo-dT pri-
mers as significantly differentially expressed in response
to nitrogen depletion, 68 did not contain an MTP
recognition site and 248 exhibited one to 18 priming
sites. For the genes with the highest number of priming
sites, the gene expression levels calculated based on the
MTP experiment were similar to the oligo-dT experi-
ment and typically yielded ratios close to one, indicating
that a higher number of exact MTP priming sites per se
did not increase detection of significant gene expression
differences.
The ratio of genes abundantly expressed to genes
meagerly expressed was largely unchanged when MTPs
were used (Table 1). Just two genes manifested a statisti-
cally significant opposite direction of differential expres-
sion in the MTP-microarray dataset compared to both
the oligo-dT dataset and the independent RNA-Seq
datasets (YDR179W-A, YKL203C). Out of 3577 genes
detected as significantly differentially expressed by
MTP-microarray, this is a very small number and may
be reasonably attributed to experimental error in the
MTP-primed reverse transcription and hybridizations.
The highest and average changes in gene expression
level were lower for MTP than for oligo-dT (Table 1).
Lastly, the gene expression level at which there was a
50% empirical probability of a significant call (GEL
50
),
illustrating the statistical power of the experiment,
demonstrated greater resolution for MTP than for oligo-
dT primers (Table 1). Three times more genes were
detected as differentially expressed in the MTP experi-
ment above its GEL
50
threshold than were differentially
expressed above the GEL
50
threshold of the experiment
performed with oligo-dT primers alone (Table 1).
Functional classification of genes differentially expressed
by S. cerevisiae grown in nitrogen-poor conditions
While meager expression in nitrogen-poor medium was
typical for genes functioning in protein synthesis and
transcription, abundant expression was typical for
genes coding for proteins involved in energy and genes
of unknown function (Figure 1a). This result was
obtained both with the aid of MTPs and with oligo-dT
primers. Using oligo-dT primers, functional categories
of metabolism, development, and energy were identi-
fied as significantly affected under nitrogen deficiency
(Fisher’sExacttest,P= 0.01, P=2.3×10
-21
,P= 0.01).
The MTP experiment identified the same functional
categories, and provided additional experimental
power, also identifying genes involved in regulation
with the environment (P=4.9×10
-5
). The classifica-
tion for which the highest number of genes was indi-
cated as significantly differentially expressed by either
approach was metabolism. Within metabolism, the
greater power of the MTP experiment provided a
stronger signal of subcategory, as more measurements
were statistically significant: 39% as indicated by MTP
and 15% as indicated by oligo-dT primers were
involved in amino acid metabolism; 36% and 15%,
respectively, in carbohydrate metabolism; and 16% and
6% in lipid metabolism.
Table 1 Overview of the results comparing use of oligo(dT) and multi-targeted primers (MTPs) for reverse
transcription
Saccharomyces cerevisiae Neurospora crassa
Feature Nitrogen depletion Protoperithecial developmenr
Oligo (dT) MTP Common Oligo (dT) MTP Common
Number of well measured genes
1
on the array 4620 6042 4573 469 416
Number of genes significantly differentially expressed (P≤0.05) 2155 3577 1839 172 406 2021
Number of up-regulated genes (P≤0.05) 1080 1855 925 66 232 31
Number of down-regulated genes (P≤0.05) 1075 1722 912 106 174 61
Highest gene expression ratio 49.15 32.6 24.26 10.74
Average gene expression ratio 2.05 1.77 1.96 1.43
GEL
502
1.51 1.27 1.91 2.08
Number of genes with gene expression ratio greater or equal to GEL50 (P≤0.05) 1237 2617 1166 102 141 52
1
well measured genes recorded after data normalization (foreground fluorescence signals were at least three standard deviations of the distributionof
intensities of the background pixels for that gene).
2
GEL
50
- gene expression level at which there is a 50% empirical probability of a significant call.
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MTPs were not biased for inference in particular path-
ways or processes, providing an even increase in power
across all of them. For nearly every pathway or process,
the microarrays performed with MTPs resulted in a
more complete assessment of expression of proteins/
enzymes building a pathway, assigning expression to
more genes than the oligo-dT-primed experiment in
each process (Table 2; see Additional File 3). Most of
the genes coding for enzymes belonging to 26 pathways
specifically related to nitrogen and amino acid metabo-
lism and biosynthesis were meagerly expressed by yeast
growing in nitrogen-poor conditions, e.g. glutamate
metabolism and urea cycle (Table 2, Figures 2, 3). An
opposite tendency of abundant expression in nitrogen-
poor conditions was observed among genes encoding
proteins composing energy-related pathways, e.g. the
citrate cycle (Figures 1 &4). Typically, enzymes catalyz-
ing reactions occurring in opposite directions exhibited
opposite regulation (Figure 2). Furthermore, we were
able to more reliably characterize the expression of mul-
tiple genes coding for isozymes, isoforms with different
cellular location and enzyme subunits (Figures 2, 3, 4).
A total of 561 genes identified as differentially expressed
by the MTP experiment and 364 genes identified as differ-
entially expressed by the oligo-dT experiment belonged to
environmental stress response genes that were inferred by
evaluating the transcriptional responses to a wide range of
stress stimuli, including nitrogen depletion [25].
RNA sequencing for assessment of transcript abundance
in S. cerevisiae under nitrogen deficiency
To validate the microarray profiling results, we performed
RNA tag sequencing with a modified Digital Gene Expres-
sion protocol that facilitated use of our custom oligonu-
cleotide primers. We sequenced 6.0 × 10
5
16-17bp tags for
yeast grown in nitrogen-rich conditions, primed with
oligo-dT, out of which 2.7 × 10
5
exactly matched a single
mRNA sequence in the S. cerevisiae genome. Similarly, we
sequenced 1.3 × 10
6
tags from nitrogen deprived yeast,
primed with oligo-dT, including 5.7 × 10
5
tags with an
exact single match. We sequenced 3.8 × 10
6
and 1.3 × 10
6
tags, respectively, from yeast grown in nitrogen-rich condi-
tions that had been MTP-primed; and 3.1 × 10
6
and 1.4 ×
10
6
, respectively, for yeast deprived of nitrogen that had
been MTP-primed. The MTP-primed sequencing yielded
larger numbers of reads in all categories. The proportion
of reads mapping to rRNAs and tRNAs was slightly
increased with MTPs, but with both oligo-dT and MTP
priming this proportion was negligible (<0.01% of all single
matches, Table 3). MTPs also did not prime a less
Figure 1 Functional classification of genes significantly differentially expressed (P≤0.05) by a) S. cerevisiae grown in nitrogen-poor
conditions and b) N. crassa protoperithecia identified by transcript profiling using oligo(dT) (red) or multi-targeted primers (blue).
Genes identified by both methods marked in green. Light-colored bars represent genes meagerly expressed and dark-colored bars represent
genes abundantly expressed. The categories are sorted by the proportion of genes meagerly- to abundantly- expressed.
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complex pool of mRNAs than do oligo-dTs. The coeffi-
cients of variation in number of tags across genes for the
oligo-dT runs were 4.3 and 3.0, and the coefficients of var-
iation for number of tags across genes for the MTP runs
were 3.3 and 3.9, so that the pool of transcriptomic tag
sequences returned when priming with MTPs were no
more rarified than the pool of transcriptomic tag
sequences returned when priming with oligo-dT.
We identified a higher number of well-measured genes
for the MTP-based dataset than for the oligo-dT data
(Table 4, see Additional File 4). In comparison to the sig-
nificantly differentially expressed gene list from microarray
profiling of yeast using oligo-dT primers, tag sequencing
confirmed 35% of the genes with gene expression levels
consistent between the two methods of measuring tran-
script abundance, but left 57% of the genes without statis-
tically significant confirmation (cf. Figures 2, 3, see
Additional File 5). In comparison to the MTP-primed
microarray experiment, MTP-based tag sequencing con-
firmed 41% of the results, leaving 48% without statistically
significant confirmation (Figures 2, 3, 4, see Additional
File 5). Differences in the functional category of metabo-
lism were validated. In particular, MTP identified 13 out
of 24 mitochondrially encoded genes as statistically signifi-
cantly differentially expressed. Oligo-dT identified none.
Oligo-dT priming led to fewer than six tags in both condi-
tions for 22 out of 24 mitochondrial ORFs. In contrast,
with MTP, only nine ORFs show fewer than six tags.
Table 2 Comparison of oligo(dT) and MTP priming in terms of number of genes significantly differentially expressed
in specific biological processes and metabolic pathways with the highest number of genes significantly differentially
expressed (P≤0.05) in S. cerevisiae and N. crassa
Process/pathway* Oligo(dT) MTP Additional genes identified by MTP Improvement (%)
Saccharomyces cerevisiae, nitrogen depletion
Carbohydrate Metabolism 136 208 72 53
Amino Acid Metabolism 114 181 67 59
Lipid Metabolism 55 89 34 62
Metabolism of Cofactors and Vitamins 43 67 24 56
Energy Metabolism 37 62 25 68
Glycan Biosynthesis and Metabolism 28 58 30 107
Xenobiotics Biodegradation and Metabolism 28 52 24 86
Nucleotide Metabolism 31 49 18 58
Signalling 19 39 20 105
Metabolism of Other Amino Acids 20 36 16 80
Cell cycle 20 32 12 60
Biosynthesis of Secondary Metabolites 9 15 6 67
Folding, Sorting and Degradation 7 12 5 71
Replication and repair 8 8 0 0
Translation 2 2 0 0
Transcription 1 1 0 0
Neurospora crassa, protoperithecial development
Carbohydrate metabolism 23 30 7 30
Translation 25 29 4 16
Amino acid metabolism 14 26 12 86
Lipid metabolism 8 19 11 138
Xenobiotics biodegradation and metabolism 5 17 12 240
Metabolism of other amino acids 3 11 8 267
Metabolism of cofactors and vitamins 3 10 7 233
Energy metabolism 9 8 -1 -11
Glycan biosynthesis and metabolism 0 5 5 **
Nucleotide metabolism 1 4 3 300
Biosynthesis of secondary metabolites 2 3 1 50
Signalling 1 1 0 0
Protein fate 0 1 1 **
Transcription 0 1 1 **
* The pathways and processes were identified using Kyoto Encyclopedia of Genes and Genomes (KEGG). For full list of S. cerevisiae pathways, see Additional File
3.
** biological process not identified by oligo(dT) primers as including any genes significantly differentially expressed by N. crassa protoperithecia.
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Figure 2 Genes significantly differentially expressed (P≤0.05) by S. cerevisiae growing in nitrogen-poor (light red/blue) and nitrogen-
rich (dark red/blue) conditions coding for enzymes involved in glutamate metabolism; identified by microarray using multi-targeted
primers (red) or oligo(dT) primers (blue). The results were validated using RNA sequencing on Illumina platform (grey; bars in the same order
as for microarray profiling). Error bars represent 95% credible intervals. NS - statistically insignificant difference (P> 0.05); * or lack of a symbol -
significant difference in gene expression level (P≤0.05); †- microarray: not well measured; tag sequencing: not detected or too few tags for a
statistical significance (P> 0.05).
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Furthermore, of those nine, five are considered dubious
ORFs anyway (see Additional File 4).
Quantitative RT PCR validation of microarray profiling
and tag sequencing of yeast grown in nitrogen-poor
conditions
To validate results of microarray and RNA sequencing
using MTP, quantitative reverse transcription PCR
(qRT-PCR) was performed (Table 5). Five genes were
selected that were well measured by the microarray-
MTP dataset but that did not produce enough signal in
the microarray-oligo-dT dataset to be measured, and
that showed the largest differences in expression
between samples. In all five of these genes, differential
expression in the direction predicted by the microarray-
MTP dataset and the RNA-seq dataset was validated.
Five genes were also selected that showed the largest,
statistically significant differences in the dataset of
microarrays primed with MTPs, but that due to lower
power showed no statistically significant difference in
the dataset of microarrays primed using oligo-dT. In
four of these five genes, differential expression in the
direction predicted by the microarray-MTP dataset and
the RNAseq dataset was confirmed. In the one excep-
tion, the qRT-PCR result contradicts the directional
trend of all four previous measurements (microarray-
MTP, microarray-oligo-dT, RNAseq-MTP, and RNAseq-
oligo-dT). Genes that were well measured in both array
experiments but that were statistically significant due to
the increased power of the MTP-based profiling had gen-
erally exhibited fairly low fold changes. Similarly low dif-
ferences in expression in the same direction were detected
by qRT-PCR, confirming the validity of MTP approach.
Microarray mRNA profiling of Neurospora crassa
undergoing sexual development
The MTP for the N. crassa was designed targeting 9846
protein-coding ORFs. Sequence VWNVNNBDKGGC
Figure 3 Genes significantly differentially expressed (P≤0.05) by S. cerevisiae growing in nitrogen-poor (light colored bar) and
nitrogen-rich (dark colored bar) conditions coding for enzymes involved in urea cycle and metabolism of amino groups; identified by
microarray using multi-targeted primers (red) or oligo(dT) primers (blue). The results were validated using RNA sequencing on Illumina
platform (grey; bars in the same order as for microarray profiling). Error bars represent 95% credible intervals. NS - statistically insignificant
difference (P> 0.05); * or lack of a symbol - significant difference (P ≤0.05); †- not well measured.
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was reverse complementary to 12-nt sequences found
one or more times in 85% of the predicted mRNAs, one
time in one tRNA, and one time in one rRNA. The
MTP was incorporated into an experiment to quantify
gene expression in mycelia and protoperithecia formed
by the fungus during growth on nitrogen-poor medium,
and the results were compared with transcript profiling
using solely oligo-dT-based reverse transcription. Appli-
cation of multi-targeted primers increased the number
of well-measured genes by 331% (Table 1, see Addi-
tional File 6). The number of genes identified as signifi-
cantly differentially expressed in N. crassa
protoperithecia rose by 136%. The highest and the aver-
age gene expression ratio estimated based on the MTP-
based experiment were lower than for data obtained
with only oligo-dT primers (Table 1). The GEL
50
value
was similar when MTPs were included, compared to
when only oligo-dT primerswereincluded.However,
38% more genes were detected as differentially
expressed in the MTP experiment above the GEL
50
threshold than were differentially expressed above
the GEL
50
threshold in the experiment primed with
oligo-dT primers alone (Table 1). Thus, more gene
expression levels were precisely estimated with MTPs,
even though in this case there was not a finer level of
resolution for small changes in gene expression.
Functional classification of genes significantly
differentially expressed by N. crassa protoperithecia
Within each of the functional groups of protein synth-
esis, transcription, energy, and genes of unknown func-
tion, a higher proportion of genes were meagerly
Figure 4 Genes significantly differentially expressed (P≤0.05) by S. cerevisiae growing in nitrogen-poor (light red/blue) and nitrogen-
rich (dark red/blue) conditions coding for enzymes involved in citrate cycle (TCA); identified by microarray using multi-targeted
primers (red) or oligo(dT) primers (blue). The results were validated using RNA sequencing on Illumina platform (grey; bars in the same order
as for microarray profiling). Error bars represent 95% credible intervals. NS - statistically insignificant difference (P> 0.05); * or lack of a symbol -
significant difference (P≤0.05); †- not well measured.
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expressed in N. crassa protoperithecia than were abun-
dantly expressed (Figure 1b). While the functional clas-
sifications with the highest number of genes significantly
differentially expressed in protoperithecia were the
“unknown”functional category or were metabolism-
related, the oligo-dT-primed experiment indicated that
genes involved in protein synthesis were significantly
affected by sexual development (Fisher’sExacttest,P=
2×10
-6
). Application of MTPs for N. crassa microarray
profiling additionally identified the functional categories
of metabolism (P= 0.003) and energy (P= 0.03) as
including a higher proportion of genes differentially
expressed in protoperithecia. Enzymes involved in fatty
acid metabolism were frequently abundantly expressed
in N. crassa protoperithecia as compared to mycelia
(Figure 5), while genes coding for ribosomal proteins
were generally meagerly expressed (see Additional File
7). Microarray hybridizations performed with MTP-
based reverse transcription identified 1 to 12 more
genes involved in each biological process than did
microarrays performed using oligo-dT primers only
(Table 2). MTPs outperformed oligo-dT primers most
strikingly in their ability to reveal differentially expressed
genes in the category of unknown and unclassified pro-
teins (Figure 1b). A gene involved in control of sexual
development, encoding clock controlled pheromone ccg-
4precursor, was among genes differentially expressed in
N. crassa protoperithecia (see Additional File 6).
Discussion
Our multi-targeted priming method features selective
targeting of multiple genomic sequences and counter-
selection against undesirable sequences. For genome-
wide gene expression assays, the addition of multi-
targeted primers offers the potential for improving the
coverage of the reverse transcription. Reverse transcrip-
tion reactions are fairly nonspecific: transcription may
be primed by non-complementary primers, other RNA
molecules present in the sample, and even by dNTPs
[8], so the priming strategy selected may be expected to
play a significant role in determining the results
obtained. For instance, commonly-used oligo-dT pri-
mers generate a high frequency of truncated cDNAs
through internal poly-A priming [26]. We demonstrated
that addition of MTP to commonly used oligo-dT
primers for yeast and N. crassa microarray profiling sub-
stantially increased the number of well measured genes
recorded after data normalization as well as the number
of genes significantly differentially expressed, compared
with the sole use of oligo-dT primers (Table 1). Addi-
tionally, MTP-based profiling manifested higher statisti-
cal power as demonstrated by lower value of GEL
50
,a
measure of empirical power to reveal differences in gene
expression [27], in the S. cerevisiae experiment.
High-throughput RNA tag sequencing validated our
findings. It confirmed the utility of MTPs, combined
with oligo-dT column purification, for priming reverse
transcription reactions, yielding an increased number of
detected and well measured genes, compared to oligo-
dT primers alone (Table 3). Priming using MTPs, we
were able to corroborate gene expression levels for a
higher number of genes than when priming with oligo-
dT (41% and 35%, respectively; Figures 2, 3, 4). To
explain this result, note that both traditional oligo-dT
and MTP priming provide biased counts and profile
only a proportion of the total RNA pool. Some genes
that are poorly primed by oligo-dT primers are those
that have protected poly-A tails due to foldback struc-
tures; other genes, such as histones in metazoan DNA,
have no poly-A tail. In a particuliarly clear case for
yeast, it has been demonstrated that many to all of the
24 putative and known mitochondrial mRNAs are not
stabilized by a poly-A tail [28-31]. For these genes,
MTPs were successful at priming and revealing gene
Table 3 Overview of transcriptomic sequence reads, comparing numbers of matches to coding ORFs, rRNAs, and
tRNAs
Single match mRNA Single match rRNA Single match tRNA Multiple matches Unmatched
Oligo-dT N-rich 266258 40 18 72849 258323
Oligo-dT N-poor 569310 38 12 99222 673334
MTP N-rich 1286862 438 18 1197105 1312253
MTP N-poor 1444282 1418 21 470558 1136358
Table 4 Overview of differential expression from
transcriptomic tag sequencing, comparing results of
oligo-dT and multi-targeted primers (MTPs) for reverse
transcription
Saccharomyces cerevisiae
Feature Nitrogen depletion
Oligo
(dT)
MTP Common
Recorded genes 4858 5078 4710
Genes significantly differentially expressed
(P≤0.05)
2272 2861 1437
Up-regulated genes (P≤0.05) 958 1437 522
Down-regulated genes (P≤0.05) 1314 1427 633
Highest gene expression ratio 166 284
Average gene expression ratio 1.84 1.90
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expression differences, whereas oligo-dT primers were
not. Some genes feature multiple sites for priming by
MTPs, leading to greater power for the detection of dif-
ferential expression for those genes. However, the effect
of multiple priming is modest, mostly increasing the
chance of a gene being well measured. The equivalent
coefficients of variation of our transcriptomic tag sequen-
cing runs indicate that the complexity of the mRNA pool
with either priming strategy is approximately the same.
The advantage of adding MTPs obtains because the
reverse transcription biases for each method of priming
are different. In combination, they exhibit greater cover-
age than either would individually. Based on our high-
throughput transcript sequencing data, MTPs might
yield slightly better coverage of expressed sequences than
oligo-dT. In terms of false negatives in the high-through-
put transcript sequencing, MTP priming exhibited a
lower false-negative rate (248 not detected by MTP mix
Table 5 Validation of microarray and RNA sequencing of yeast grown in nitrogen poor (MM) and nitrogen rich (SC)
conditions using Real Time RT PCR
Microarray RNA sequencing RT PCR
Gene ID MTP oligo(dT) MTP oligo(dT) MTP Oligo(dT)
Ratio MM/SC p value Ratio MM/SC p value Ratio MM/SC p value Ratio MM/SC p value Ratio MM/SC Ratio MM/SC
YDL244W 1.98 0.00 3.16 0.11 28.94 39.62
YGR225W 1.72 0.00 7.65 0.00 4.08 3.13
YGR249W 1.66 0.01 5.80 0.00 4.79 8.20
YMR025W 1.51 0.00 3.96 0.15 2.10 1.57
YNR072W -1.24 0.05 -6.32 0.00 -2.34 -1.36
YFR034C -1.42 0.02 -1.25 0.10 -9.08 0.00 -6.41 0.08 1.30 1.24
YKR053C -5.37 0.00 -1.88 0.14 -2.16 0.00 1.15 0.51 -2.10 -2.05
YLR126C 1.54 0.00 1.36 0.11 5.54 0.05 1.40 0.42 1.58 1.95
YMR003W 1.25 0.01 1.11 0.26 7.91 0.01 2.65 0.08 1.03 1.95
YOR252W 1.29 0.01 1.03 0.46 6.33 0.00 1.54 0.10 1.99 1.54
Figure 5 Genes significantly differentially expressed (P≤0.05) by N. crassa protoperithecia (light red/blue) and mycelium (dark red/
blue) coding for enzymes involved in fatty acid metabolism; identified by microarray using multi-targeted primers (red) or oligo(dT)
primers (blue). Error bars represent 95% credible intervals. NS - statistically insignificant difference (P> 0.05); * or lack of a symbol - significant
difference (P≤0.05); †- gene not well measured.
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that were detected by oligo-dT alone) than did oligo-dT
priming alone (368 not detected by oligo-dT that were
detected by MTP). Correlations between replicate tag
sequencing results and between replicate MTP-based
microarray results were similar. Next generation RNA
tag sequencing offers a promise of increasing power,
depth, and sensitivity of gene expression analysis, but
even a limited depth of RNA sequencing remains costly
compared to a highly replicated experiment using micro-
arrays generated in the lab.
Finally, qRT-PCR validated the results based on MTP-
priming. It confirmed that genes identified as well mea-
sured or as significantly differentially expressed only by
MTP-primed microarray, had changed in expression in
the direction revealed by the MTP-primed experiments,
regardless of the primer combination used. This obser-
vation suggests that incorporating MTPs increases the
power for detection of differential expression by micro-
arrays, by increasing the coverage of the priming of
reverse transcription while maintaining low noise due to
nonspecific priming. Logistic regressions showed some
evidence that genes with more priming sites were mea-
sured with increased power with MTPs applied than
genes with fewer MTP priming sites, but this observa-
tionwastrue(albeittoalesserextent)foroligo-dT
priming as well. This commonality may well arise
because longer genes have more MTP sites and are bet-
ter measured with either MTPs or oligo-dT primers.
Considering the 4762 observations underlying these
effects, their statistical significance is unsurprising. The
effect sizes are not large. It would be possible to proce-
durally minimize the variation in the number of MTP
sites, but effectively, this minimization of variation
would entail searching for MTPs that were less powerful
for some genes. That would be counter-productive to
the effort to measure genome-wide gene expression for
as many genes as possible to the best precision possible.
Because such gene-specific effects exist at approximately
the same levels no matter the method of priming,
decent statistical approaches toward microarray data
have featured effects estimated for each gene, thus pro-
viding P-values and confidence or credible intervals that
appropriately reflect the variation in power across genes.
Markedly successful enhancement of the power of tran-
scriptomic approaches was achieved here by usage of
MTPs subsequent to a crude filtration provided by poly-
A+ purification columns, which typically bring the RNA
content to approximately 50% poly-A+ RNA and 50%
other RNA [32,33]. An alternate approach with potential
to further increase power would be to forego any col-
umn purification of poly-A+, relying solely on MTPs to
pick out messenger RNA from other species of RNA.
Functionally, inclusion of MTPs provided a more
complete picture of cellular processes undergoing
change during fungal growth in nitrogen deficient con-
ditions and in early sexual development. Interestingly,
the most striking difference in the ability to detect genes
differentially expressed between MTPs and oligo-dT pri-
mers was observed among N. crassa unknown and
unclassified genes (Figure 1b). Genes expressed at a low
level have a lower probability of being identified and
characterized than those expressed at a higher level [34].
Only 42% of genes in N. crassa genome have been
assigned known functions [35] so identifying their
expression signatures and inferring functional classifica-
tion are key objectives of future experimentation [36].
The increased levels of yeast transcripts coding for
enzymes involved in the TCA cycle observed here
(Figure 4) supports a finding that the content of TCA
cycle compounds increases during yeast starvation [37].
Both carbon and nitrogen limitation have been shown
to enhance respiration relative to fermentation in yeast
[21,38]. Amino acid metabolism was strongly affected by
exposure to nitrogen deficiency, as in the filamentous
fungus Magnaporthe grisea [17]. Prevalent down-regula-
tion of glutamate metabolism pathway (Figure 2) was in
accordance with decline of total glutathione pool found
in nitrogen-starved S. cerevisiae [39]. Similarly, the urea
cycle was down-regulated, with an exception of the
branch responsible for urea degradation (Figure 3). Urea
amidolyase, the enzyme corresponding to that branch,
contains both urea carboxylase and allophanate hydro-
lase activities. It also converts allantoin to ammonia and
carbon dioxide, enabling S. cerevisiae to use allantoin as
a sole nitrogen source [40]. The urea cycle operates in
terrestrial animals to detoxify ammonia, and in yeast
plays a principal role in the biosynthesis of arginine.
Within these pathways, we were able to characterize
expression of multiple isozymes and isoforms (Figures 2,
3, 4) providing a key component of a full understanding
of expression regulation in nitrogen-deficient medium.
The general trend of shutting down amino acid biosynth-
esis and metabolism appears essential for survival during
prolonged periods of starvation. These results from our
experiments are entirely consistent with a functional
category analysis of independent data from the unrepli-
cated time-course experiment by Gasch et al. [25].
A number of genes abundantly expressed by yeast
during growth in nitrogen-poor conditions are compo-
nents of the environmental stress response that was
characterized by evaluating the transcriptional responses
to a wide range of stress stimuli, including nitrogen
depletion [25]. The remaininggenesmightbeinvolved
in immediate responses specific to nitrogen limitation,
but many are likely associated with transitive effects that
occur over time, such as successive nutrient depletion,
pH change, cell density, and growth arrest. The finding
that development-related genes were significantly
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affected in our study may be related to growth arrest or
to a morphological switch from the yeast to a filamen-
tous or pseudohyphal growth triggered by nitrogen star-
vation in the presence of a fermentable carbon source,
such as glucose [41]. Nutrient and nitrogen sensing
plays important roles in fungal development in general,
and specifically in critical aspects of pathogenicity and
virulence, for both animal and plant pathogens.
Dimorphic pathogens such as the phytopathogenic smut
fungi, Ustilago maydis and Microbotryum violaceum,
must switch from a yeast-like to a filamentous form in
order to cause disease [42].
N. crassa protoperithecia consist of a small knot of
vegetative hyphae surrounding ascogonial cells. Their
formation requires extensive cell proliferation. Thus, it
was not surprising to find a large proportion of differ-
entially expressed genes involved in metabolism, espe-
cially carbohydrate metabolism (Table 2), providing
energy and the robust structural components necessary
for specialized cell formation. The shift in transcript
accumulation of genes involved in fatty acid metabo-
lism (Figure 5) supports previous observations that the
fatty acid composition of sexual tissues of Neurospora
differ substantially from the composition of asexual tis-
sues. For instance, mutations in the gene encoding the
b-fatty acid synthase affect sexual development [43].
On the other hand, we expected to identify known
developmental genes responsible for the formation of
protoperithecial reproductive structures, but the num-
ber of genes significantly differentially expressed
involved in the functional category of development was
very low. One of them coded for clock controlled
pheromone ccg-4 precursor which had been first iden-
tifiedasagenethatisexpressedwitha22hrhythm
under the control of the circadian biological clock
[44]. The expression of the pheromone precursor
genes is mating-type specific and is under the control
of the mating type locus. These genes are highly
expressed in conidia and under conditions that favor
sexual development [45].
We found a reduced level of transcripts related to pro-
tein synthesis (Figure 1, see Additional File 7) in both
S. cerevisiae under nitrogen starvation and in N. crassa
sexual development occurring as a developmental
response to nitrogen depletion. Ribosome biogenesis
and protein translation are among the most energy-con-
suming cellular processes [46]. Thus, these pathways are
tightly controlled upon nutrient limitation. Decreasing
levels of protein, RNA and soluble aminonitrogen were
observed during protoperithecia development as early as
1975 [47]. Additionally, ribosomes are rapidly degraded
by autophagy upon nutrient starvation in S. cerevisiae,
implying that degradation of excessive ribosomes may
help to shut down protein translation rapidly and
provide an important source of new building blocks to
maintain cellular homeostasis [48].
The MTPs used in this study were designed to prime
76-85% of mRNAs rather than all the genes, so that
reverse transcription of rRNA and tRNA could be
simultaneously minimized. An additional step of primer
design could be conducted to specifically target the
remaining sequences to eliminate or further diminish
the need to rely on low-complexity, low-specificity
oligo-dT primers. For transcript profiling in prokaryotes,
such a tiered approach would constitute an appealing
alternative to the use of non-specific random primers.
Correspondingly, the modification of Illumina massively
parallel RNA tag sequencing presented here, involving
the use of custom primers, presents the possibility of
performing Digital Gene Expression assays on transcrip-
tomes lacking poly(A) tails or on a designable fraction
of all the genes. Furthermore, future applications of
multi-targeted primers may include concurrent selective
amplification of DNA sequences of interest from one or
multiple genomes for the high-throughput sequencing
of multiple homologous loci. Locked nucleic acid
nucleotides could be included in the MTP sequence to
increase binding strength and specificity, and to
decrease non-specific amplification [49].
Conclusions
Here we presented a novel priming strategy for genome-
wide gene expression assays, featuring selective targeting
of mRNA and counter-selection against rRNA and
tRNA. We demonstrated superior performance of two
MTPs compared to oligo-dT by microarray profiling of
the response of Saccharomyces cerevisiae to nitrogen
deficiency and by profiling Neurospora crassa early sex-
ual development. MTPs resulted in higher sensitivity,
yielding more well measured genes after data normaliza-
tion, more genes significantly differentially expressed,
and a greater power to detect meager differences in
gene expression. Our results provide the most complete
and detailed expression profiles of the nitrogen starva-
tion response to date. Future applications of multi-tar-
geted primers may include concurrent selective
amplification of DNA sequences of interest from one or
multiple genomes for the high-throughput sequencing
of multiple homologous loci.
Methods
Multi-targeted primers (MTPs)
Sequences for all three types of RNA were obtained
from the Saccharomyces Genome Database [50] and the
Neurospora crassa Genome Database [35]. To identify a
degenerate sequence that occurs in a maximal number
of mRNAs and in a minimal number of tRNAs and
rRNAs, exhaustive search is not a feasible option as
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there are 16
12
such degenerate sequences. Our heuristic
search started with a random degenerate 12-mer oligo-
nucleotide. Iteratively, this oligonucleotide was randomly
mutated and a score was computed as m/(m
t
*(1+t+
r)),wherem,t,andrare the number of hits of the
degenerate sequence to the annotated mRNAs, tRNAs,
and rRNAs, respectively, and m
t
is the total number of
annotated mRNAs. If a randomly generated number (0-
1) was smaller than the ratio of the new score to the
previous, raised to the power of ten, then the new oligo-
nucleotide was kept. Otherwise, it was discarded. This
procedure was iterated so that the oligonucleotide
experienced 2*10
6
mutations before the search was ter-
minated. To ensure convergence upon a globally opti-
mal priming sequence, several different random initial
sequences were used.
Each new oligonucleotide obtained was recorded.
From the ranked list, we selected a high-scoring
sequence that additionally showed strong binding
(GC) in the 3′end. The MTP primer was designed
to be reverse-complementary to the selected
dodecanucleotide.
Yeast strain and conditions
Saccharomyces cerevisiae S288c was grown in synthetic
complete medium (SC) at 30°C shaking at 160 rpm until
mid log phase (OD
600
= 0.4). For nitrogen depletion,
cells were collected and resuspended in an equal volume
of minimal medium without amino acids or adenine and
with limiting concentrations (0.025%) of ammonium sul-
fate. Cultures were harvested after 12 h incubation, flash
frozen in liquid nitrogen and stored at -80°C [25].
Sample preparation and hybridization to yeast
microarrays
A set of clones containing 6,188 verified open reading
frames (ORFs) from the Saccharomyces Genome Project
was amplified by PCR and the DNA was spotted on
CMT-GAPS g-aminopolysilane-coated glass slides
(Corning, Corning, NY). RNA was extracted and cDNA
synthesized as in Townsend et al. [51]. Briefly, two μgof
purified mRNA were reverse transcribed using the
reverse transcriptase Superscript II (Invitrogen, Carls-
bad, CA) for 2 h at 42 C. The reaction was primed with
either 0.5 μg oligo-dT primers or 0.25 μgoligo-dT
mixed with 0.25 μg of yeast-specific MTP. Amino-allyl-
dUTP (Sigma) was incorporated into cDNA along with
dNTPs. The cDNA was labeled reciprocally with cyanine
dyes and used for hybridization as Townsend et al. [51].
The cDNA from yeast cultured in nitrogen-rich and the
cDNA from yeast cultured in nitrogen-depleted medium
were reciprocally labeled and competitively hybridized.
All replicates originated from independent reverse tran-
scription reactions.
Data acquisition and analysis
Hybridized microarrays were scanned and gridded with
a GenePix 4000B microarray scanner (Axon Instru-
ments, Foster City, CA), normalized by background-sub-
tracted mean-by-mean normalization of well measured
spots as in [51,52], and statistically analyzed using a
Bayesian analysis of gene expression levels (BAGEL
[53]). Fluorescence intensity values were adjusted by sub-
tracting background from foreground. A gene was con-
sidered well measured if the foreground fluorescence
signals were higher than three standard deviations of the
distribution of intensities of the background pixels for
that gene. Genes were deemed significantly differentially
expressed when P≤0.05. To assess the power of the
experiments to detect smaller differences in gene expres-
sion, the gene expression level at which there was a 50%
empirical probability of a significant call (GEL
50
)was
inferred by logistic regression of statistical significance
against fold-change as in Townsend [27]. Functional
annotation was performed based on the Saccharomyces
Genome Database [50] and Kyoto Encyclopedia of Genes
and Genomes (KEGG) [54]. Significance of abundantly
differentially expressed functional categories was calcu-
lated using a Fisher’s exact test. Raw expression data and
analysis results are deposited under accession #25 at the
Filamentous Fungal Gene Expression Database (FFGED)
[36,55] and under accession GSE230003 at the NCBI
Gene Expression Omnibus (GEO) database [56].
Transcriptional tag sequencing of yeast in nitrogen-poor
conditions
Digital Gene Expression (DGE) measurement was per-
formed using Tag Profiling with DpnII Sample Prep Kit
(Illumina, San Diego, CA). Oligo-dT priming was per-
formed as instructed using 2.5 μg of the same total RNA
that was extracted from S. cerevisiae for microarrays. For
multi-targeted priming, mRNA was purified on Oligo-dT
Cellulose Columns (Molecular Research Center, Cincin-
nati, OH) and 2 μg were used for reverse transcription
with 0.5 μg5′-amine yeast-specific MTP and 5 mC dNTP
10 mM each (Illumina). The resulting first strand cDNA
was cleaned with Microcon-30 microconcentrators and
coupled to Sera-Mag® magnetic carboxylate microparti-
cles (Seradyn, Indianapolis, IN). cDNAs was immediately
subjected to second strand synthesis and sequencing
library preparation with theIlluminakit.Sampleswere
then sequenced on an Illumina Genome Analyzer II. Raw
sequence reads of transcript tags are deposited under
accession #67 at the FFGED [36,55].
Transcript tag analysis
The DGE protocol used generates 16-17 nt tags ligated
to adapters. RNA sequencing produced reads of 28 bp.
After discarding the included adapter sequence, we
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searched for perfect matches of the tag sequence in the
S cerevisiae genome. Statistical significance was calcu-
lated by estimating the proportion pof tags per gene by
maximum likelihood (maximizing p
n
(1-p)
N-n
where nis
the number of tags observed for the gene and Nis the
total number of tags for all genes), and comparing the
likelihood of the observed numbers of tags with a single
maximum likelihood proportion for both samples (p)to
the likelihood of the observed numbers of tags with two
independent maximum likelihood proportions (p
1
and
p
2
, one for each sample). To estimate P-values, negative
two times the log likelihood ratio was compared to a
chi-square distribution with one degree of freedom. Sta-
tistical results for transcript tag analysis are deposited
under accession #67 at the FFGED [36,55].
Real Time RT-PCR validation of yeast microarray and RNA
sequencing results
To validate results of yeast microarrays and RNA
sequencing, ten genes were selected whose expression
was well measured in the MTP-based experiment, but
not well measured or insignificant in the oligo-dT
experiment. An equal amount (2 μg) of mRNA, as had
been used for microarray profiling, was reverse tran-
scribed with Superscript II reverse transcriptase (Invitro-
gen, Carlsbad, CA) and 0.25 μg oligo-dT mixed with
0.25 μg yeast-specific MTP or 0.5 μg oligo-dT. Relative
transcript abundance was measured using gene specific
primers (see Additional File 8) and SYBR Green PCR
Master Mix (Applied Biosystems, Carlsbad, CA). The
reaction was run on Applied Biosystems 7500 Fast Real-
Time PCR System according to manufacturer’s recom-
mendations. A gene coding for mitochondrial ribosomal
protein of the small subunit (YDR175C), whose expres-
sion did not change in the microarray experiment, was
used as endogenous control. Transcript levels were cal-
culated from triplicates within one plate using the com-
parative C
T
(ΔΔC
T
)method(ABIapplicationnotes,
Guide to Performing Relative Quantitation of Gene
Expression Using Real-Time Quantitative PCR).
Induction of Neurospora crassa sexual development
Neurospora crassa (mat A) obtained from Fungal Genet-
ics Stock Center (strain 2489) was grown on nitrogen-
poor synthetic cross medium (SCM) agar plates with
1.5% sucrose as carbon source, at room temperature
under natural light. The mycelium was flash frozen by
application of liquid nitrogen, then harvested after 18
hours growth. Protoperitheciaweresimilarlyharvested,
after 6 days.
Transcript profiling of Neurospora crassa protoperithecia
Total RNA was extracted from homogenized frozen tis-
sue and mRNA was purified with TRI REAGENT and
Oligo-dT Cellulose Columns (Molecular Research Cen-
ter). Reverse transcription, data acquisition and analysis
were conducted as for S. cerevisiae. The cDNAs were
hybridized [57] to whole-genome-spotted 70-mer N.
crassa microarrays [58]. Samples from N. crassa mycelia
and protoperithecia were competitively hybridized. Each
experiment consisted of a dye-swap and an additional
replicate hybridization. Functional classification was
based on MIPS Neurospora crassa Genome Database
[52] and KEGG [54]. Raw expression data and analysis
results are deposited under accession #2 at the FFGED
[36,55] and under accession GSE23003 at the NCBI
GEO database [56].
Additional material
Additional File 1: Number of MTP priming sites in S. cerevisiae
genes. Table of the number of MTP priming sites in S. cerevisiae genes
Additional File 2: Microarray profiling results comparing S.
cerevisiae grown in nitrogen rich (SC) or poor (MM) conditions
using oligo(dT) and multi-targeted primers (MTPs) for reverse
transcription. Table of microarray profiling results comparing S. cerevisiae
grown in nitrogen rich (SC) or poor (MM) conditions using oligo(dT) and
multi-targeted primers (MTPs) for reverse transcription. For each priming
method for each gene, relative gene expression levels in SC and MM, the
additions and subtractions for those levels to demarcate 95% credible
intervals, and the p-value for the direction of difference observed are
reported.
Additional File 3: Comparison of oligo(dT) and MTP priming in
terms of number of genes significantly differentially expressed in
specific biological processes and metabolic pathways. Table listing
biological processes and metabolic pathways with the highest number
of genes significantly differentially expressed (P ≤0.05) in S. cerevisiae
and N. crassa, the number of genes differentially expressed as detected
by oligo(dT)-primed and MTP-primed reverse transcription and
microarray hybridization, the number of additional genes identified by
MTP, and the percent improvement by MTP.
Additional File 4: Number of aligned reads recorded for each gene
with oligo(dT)-primed and MTP-primed RNA tag sequencing of S.
cerevisiae grown in nitrogen rich (SC) or poor (MM) conditions.
Table listing each ORF, the number of oligo(dT) primed reads aligning
from nitrogen poor (MM) conditions, the number of oligo(dT) primed
reads aligning from nitrogen rich (SC) conditions, the number of multi-
target primed reads aligning from nitrogen poor (MM) conditions, and
the number of multi-target primed reads aligning from nitrogen rich (SC)
conditions
Additional File 5: Comparison of transcript profiling of S. cerevisiae
grown in nitrogen rich (SC) or poor (MM) medium obtained with
microarray or RNA tag sequencing using MTP or oligo(dT) primers.
Table listing S. cerevisiae ORFs and results of both microarray and RNA
tag sequencing by both MTP and oligo(dT) priming. Results listed for
each platform and priming methodology are the fold change of MM/SC
and the p-value.
Additional File 6: Microarray profiling results for N. crassa
undergoing sexual development when multi-targeted primers or
oligo(dT) primers were used for reverse transcription. Table listing
the Broad ID and MIPS ID of well-measured genes in N. crassa
protoperithecia (PP) and mycelium (Myc), relative expression levels,
additions and subtractions for 95% credible intervals, and p-value, when
multi-targeted primers (MTP) or oligo(dT) primers were used for reverse
transcription.
Additional File 7: Comparison of expression levels of ribosomal
genes in Neurospora crassa. Table of ribosomal genes and their fold
change in expression in protoperithecia over mycelium, as measured by
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two-color microarray hybridization of oligo(dT)-primed cDNA and multi-
target-primed cDNA.
Additional File 8: Sequences of primers used for Real Time RT-PCR.
Table listing the gene measured, and the forward primer and reverse
primer used.
Acknowledgements
This work was supported by funding from Yale University.
Author details
1
Department of Ecology and Evolutionary Biology, Yale University, 165
Prospect St, New Haven, CT 06511, USA.
2
Department of Ecology and
Evolutionary Biology, Program in Computational Biology and Bioinformatics,
and Microbiology Graduate Program, Yale University, 165 Prospect St, New
Haven, CT 06511, USA.
Authors’contributions
JPT, AA, and TAC conceived and designed the experiments. JPT and FLP
designed and developed the MTP algorithm. AA and TAC performed the
microarray profiling. AA performed RNA tag profiling. ZW performed Real
Time PCR validations. AA, JPT and FLP analyzed the data. AA and JPT wrote
the manuscript. All authors read and approved the final manuscript.
Received: 25 June 2010 Accepted: 17 August 2010
Published: 17 August 2010
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doi:10.1186/1471-2164-11-477
Cite this article as: Adomas et al.: Multi-targeted priming for genome-
wide gene expression assays. BMC Genomics 2010 11:477.
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