Nucleic Acids Research Advance Access published online on April 8, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn165
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
MultiPriDe: automated batch development of quantitative real-time PCR primers
A. C. Ziesel1,2,*,
M. A. Chrenek1,2 and
P. W. Wong1
1Department of Ophthalmology, Emory University, B5500 Clinic B, 1365 Clifton Road NE, Atlanta, GA 30322, USA and 2Department of Biological Sciences, University of Alberta, CW405 Biological Sciences Centre, Edmonton, Alberta T6G 2E9, Canada
*To whom correspondence should be addressed. Tel: +1 404 778 5531; Fax: +1 404 778 4411; Email: aziesel{at}emory.edu
Received January 1, 2008. Revised March 21, 2008. Accepted March 24, 2008.
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ABSTRACT
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Quantitative reverse transcriptase polymerase chain reaction
(qRT–PCR) is a commonly employed gene expression quantification
technique. This requires the development of appropriately targeted
oligonucleotide primers, which necessitates the identification
of ideal amplicons, development of optimized oligonucleotide
sequences under most favorable pre-determined reaction conditions,
and management of the resultant target-oligonucleotide pair
information for each gene to be studied. The Primer3 utility
exists for development of oligonucleotide primers and fills
that role effectively. However, the manual process of identifying
target sites and individually generating primers is inefficient
and prone to user-introduced error, especially when a large
number of genes are to be examined. We have developed MultiPriDe
(Multiple Primer Design), a Perl utility that accepts batch
lists of Gene database identifiers, collects available intron
and exon position data critical to qRT–PCR primer development,
and supplies these sites as identified targets for the Primer3
utility. This automated ‘gene to primer’ procedure
is coupled with a set of optimized hybridization conditions
used by the Primer3 utility to maximize successful primer design.
MultiPriDe and assembled repeat libraries are available upon
request. Please direct requests to
aziesel{at}emory.edu.
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INTRODUCTION
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Quantitative reverse transcriptase polymerase chain reaction
(qRT–PCR) is a technique by which expression levels of
individual genes can be quantified by measuring the abundance
of their mRNA products relative to a standard (such as 18S rRNA).
It represents a robust means of gene expression quantification
on its own, and is an ideal companion technique for confirmation
or further inspection of results obtained by methods such as
SAGE (
1) and microarray analysis strategies (
2). The basis of
qRT–PCR is traditional RT–PCR, cycling temperatures
to allow repeated steps of double-stranded nucleic acid melting,
primer annealing, polymerase-driven synthesis of new double-stranded
nucleic acid. In qRT–PCR, oligonucleotide primers are
designed to span intronic sequences, the portions of the genome
not represented in mature mRNA products. When these primers
bracket an intronic splice site, a small product will be produced
when the primers anneal to mRNA during the reaction. An easily
recognizable larger product (or in the case of an extraordinarily
long intron, no product at all) would be produced should any
contaminant genomic DNA persist in the sample used for the reaction
(
Figure 1). One such detection stratagem, referred to here as
SYBR Green, employs a chemical agent that binds to synthesized
double-stranded nucleic acid products generated during the course
of the qRT–PCR (
3). This associated molecule then fluoresces
at 520 nm when excited with a wavelength of 497 nm; this emitted
wavelength is detected by the qRT–PCR apparatus and recorded
over the course of the reaction. Combining information regarding
increased fluorescent emission, temperature cycling, initial
amounts of RNA used in the qRT–PCR reaction and fluorescence
emission of an included standard allows a calculation to be
made determining the levels of the targeted RNA species, quantifying
its expression in the original sample.
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Figure 1. RT–PCR primers are chosen in adjacent exons, with the predicted product spanning an intronic splice site. In this way, should any genomic DNA persist in the RNA preparation, any amplification from genomic DNA will produce a long product (A), containing both the targeted sequence and intervening intron. Amplification from mRNA (B) will produce only a short product as the intronic sequence has been spliced out of the mRNA.
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Determining appropriate intron-spanning target sites and developing
oligonucleotide primers against them involves several steps,
starting with identifying target genes, determining their genomic
sequence, their mRNA coding sequence (CDS) and the location
of intervening intronic sequences. Oligonucleotide primer development
can be done by manually identifying ideal primer sites, but
the majority of primer design is currently conducted with the
aid of computational tools, such as MuPlex (
4), PriFi (
5), the
widely adopted Primer3 utility (
6) or its alternate interface,
Primer3Plus (
7). Primer3 is freely available as a standalone
package and is available online through a simple web interface.
However, for those users disinclined to run Primer3 locally
through a command line interface, designing primers against
identified targets for each chosen gene individually through
a web interface is potentially time-consuming and requires careful
management of retrieved information. In addition to the management
of these aforementioned data, there is also the issue of developing
targets and primers, and selecting reaction conditions that
will be most optimally successful. The difficulty of coordinating
these data increases with the size of the qRT–PCR project
undertaken. We elected to address these issues of target site
identification, primer development and optimization, and data
organization to ameliorate the process for molecular biology
laboratories. To this end, we have developed a set of optimized
qRT–PCR primer design conditions and a script that incorporates
these conditions into an automated design procedure.
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METHODS
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MultiPriDe is designed to streamline and standardize the process
starting with target gene identification through to primer design.
After user identification of targets, MultiPriDe handles the
entire procedure independent of additional user input, and produces
organized, easily accessible primer design information. Moreover,
as all primer sets are designed with the same optimized reaction
conditions, all qRT–PCR reactions should be performable
simultaneously or with the same settings from run to run. The
steps that MultiPriDe follows are illustrated in
Figure 2: starting
with initial input, the script identifies and retrieves the
required information from NCBI resources (
8). It then determines
intronic splice sites, which are ideal qRT–PCR target
sequences. These ideal targets are then directed to a local
install of the Primer3 package that uses our empirically optimized
hybridization conditions to develop sets of primers amplifying
each identified target. The identified CDS, intronic splice
sites and primer design results are available to the user upon
completion. We have chosen to employ a locally installed version
of Primer3 for use with MultiPriDe chiefly to allow users to
exploit the power of their own laboratory computers rather than
further taxing existing publicly available Primer3 servers.
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Figure 2. The process MultiPriDe follows, starting with inputted GeneIDs and concluding with identified CDS and primers for each intronic splice site. The processes described in the central gray box are those that are performed by the user's computer.
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We developed a specific set of parameters for oligonucleotides
designed by MultiPriDe, specifically deviating at key points
from those default parameters specified by the Primer3 package.
First, we chose an annealing temperature range of 55.0°C
(minimum), 57.5° (optimum) and 60.0°C (maximum) instead
of the default 60.0, 63.0 and 65.0°C range specified in
the Primer3 defaults. We chose this range because it typically
led to acceptable qRT–PCR reactions when working with
mouse and rat RNA samples, and because oligonucleotides designed
with our temperature range typically exhibited a narrower range
of ideal primer concentrations in reaction (unpublished results).
We also adjusted the maximum target oligonucleotide size to
25 nt from 27 nt to compensate for the reduction in annealing
temperature. We altered the default settings for maximum self-complementarity
and maximum 3' self-complementarity from values of 8.00 and
3.00, respectively, to 5.00 and 2.00, respectively, to reduce
primer dimer formation. Finally, we use a specialized repeat
library to develop the qRT–PCR primer sets described here.
This repeat library is a merger of two of the base libraries
offered with the Primer3 package (the ‘rodent and simple’
and ‘human’ libraries). We use this merged library
for ease of primer development in our own cross-species experiments
and it is not intended to confer any special benefit with the
exception of simplicity for multi-species batch primer design.
Use of our merged library is not necessary with MultiPriDe.
MultiPriDe was written entirely in Perl, and uses the LWP module for certain functions. Local installations of standard Perl 5.8.0, the LWP module [available through the Comprehensive Perl Archive Network (CPAN)] and the Primer3 package (available at http://www-genome.wi.mit.edu/genome_software/other/primer3.html) are required for functionality. The user supplies an individual or a batch list of GeneID numbers via command line interface. MultiPriDe first queries NCBI, accessing the Entrez Gene records for these numbers. The script then collects the genomic sequence entry and first join data entry for that record. This information is used to identify exons and intronic splice sites in the genomic sequence, and prints the compiled CDS to the screen, along with CDS nucleotide positions for intronic splice sites. These splice sites will be specified as targets for Primer3 oligonucleotide design in subsequent steps. In the case of genes that are composed of a single exon, no target is specified; the entire monoexonic CDS is a potential target site for oligonucleotide design. Next, MultiPriDe invokes a locally installed version of Primer3, and using our hybridization conditions coupled with the intronic splice site positions, develops a maximum of 100 primer pairs spanning each splice site in the CDS. MultiPriDe creates directories named for each GeneID queried, and writes into these the Primer3 output files named according to the convention GeneID_splicesite.pr3. These .pr3 files are readable in any text editor and are equivalent to the output a user may receive when performing an equivalent analysis using a Primer3 server. Those GeneIDs that lack any of the requisite information (an entry, genomic sequence or join data) return an error message describing the nature of the error and an output file containing FASTA-formatted cDNA sequence if available.
An example of our own use of MultiPriDe follows. Cell cultures were prepared of human WERI-Rb1 (gift of Dr J. Boatright, Emory University, Atlanta, Georgia) or rat NRK-52E cells [American Type Culture Collection (ATCC), Manassas, VA, USA] for subsequent RNA extraction. Dishes (60 mm) of cells were grown to monolayer, media was removed and 1 ml of Trizol (Invitrogen, Carlsbad, CA, USA) was added to each dish. The dishes were shaken at 80 r.p.m. for 2 min, then 900 µl of lysed cells were removed from the dish. For mouse RNA, a mixture of RNA extracted from multiple tissues was used, including brain, heart, mammary gland, muscle, liver, lung, ovaries, spleen, submandibular gland and uterus. RNA was prepared from both cultured cells and mouse tissue using Trizol according to the manufacturer's recommended protocol with the inclusion of an additional chloroform wash following organic phase separation, quantified using fluorescence methods and then diluted to 100 ng/µl. RNA samples weighing 5 µg each were treated with Turbo DNA-free DNase (Ambion, Austin, TX, USA) to remove any contaminating DNA. DNase-treated RNA was then purified and reconcentrated using Qiagen RNeasy MinElute columns (Qiagen, Valencia, CA, USA) and RNA was eluted in 100 µl of nuclease-free water (2 ng/µl).
Oligonucleotides designed using MultiPriDe were ordered from (MWG Biotech, High Point, NC, USA) as high-purity salt free in 0.01 µmol size. Each oligonucleotide was dissolved in nuclease-free water to 100 µM. Two forward and two reverse primers from the list of primers identified from our MultiPriDe analysis were ordered for a single splice site for each gene, generating four unique pairs of primers for each gene. For a given multiexonic gene, primer pairs were chosen from MultiPriDe's generated results with priority placed on (i) splice site proximity to 5' end of transcript, (ii) splice site at least 100 bp from either end of transcript and (iii) at least two pairs of primers were generated for the specific chosen splice site. Individual oligonucleotides were aliquoted into pairs at 1 µM concentration (per primer) with the pairs set up as follows: pair 1—F1/R1; pair 2—F1/R2; pair 3—F2/R1; pair 4—F2/R2. This concentration was considered a 5 x primer pair stock for subsequent testing of primers at an initial 200 nM concentration of each primer in qRT–PCR. For reactions that showed a single dissociation peak in the reactions when template RNA was added but showed primer dimers in the absence of template RNA, primers were retested at 100 nM concentration. For reactions that showed no amplification of fluorescent signal in the presence of template the primers were retested at 400 nM.
qRT–PCRs were performed using a QuantiTect SYBR green one-step RT–PCR kit (Qiagen). Reactions (25 µl) were set up with 5 ng of template RNA or an equivalent volume of nuclease-free water as a control and 100, 200 or 400 nM concentration of each of the primers in a pair to be tested. qRT–PCRs were performed in an ABI-7500 real-time thermocycler (Applied Biosystems, Foster City, CA, USA). The reverse-transcriptase reaction began with 15 min at 95°C to activate the polymerase, 50°C for 30 min and then followed with 40 cycles of 95°C for 15 s (strand dissociation), 55 to 60°C for 30 s (primer annealing, temperature dependent on the primer pair), and 72°C for 40 s (DNA synthesis). Fluorescence data was collected during the extension step of each cycle. Annealing temperature was 55°C for primers designed to have a melting temperature of 57.5°C, and 60°C for primers designed to have a melting temperature of 63°C. Amplification curves were generated using the supplied Applied Biosystems SDS v1.2 software. Following qRT–PCR, dissociation curves were generated for each sample. The qRT–PCR products were dissociated at 95°C for 5 min, then reassociated at 60°C. Incremental steps of +0.1°C were done with fluorescence data collected at each step and the derivative of the change in fluorescence was plotted by the SDS software.
Amplification plots with and without template RNA were examined for each primer pair. Those that showed high efficiency amplification of signal with template added and no amplification of signal in the absence of template were considered good amplification plots (Figure 3). A primer pair was considered to have failed if the amplification plot showed amplification of signal from the reaction that contained no template or if the efficiency of the amplification was weak in the presence of template. Dissociation curves with and without template RNA were examined for each primer pair. Those that showed a single peak for the reaction containing template and no peaks for the reaction containing no template were considered to be good dissociation curves; we consider our threshold for success to be quite stringent, and may be more stringent than is required for some purposes (Figure 3). Those that showed multiple peaks for the reaction containing template or any peak including low melting temperature peaks (primer dimers) for the no template reaction were considered to be bad dissociation curves. Each primer pair was graded as passed if both the amplification plot and the dissociation curves were good, or failed if either the amplification plot or the dissociation curve was bad, according to our aforedescribed criteria.
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Figure 3. A sample RT–PCR using MultiPriDe-developed primers. (A) Demonstrates an amplification curve for these primers, which is a plot of fluorescence intensity on the ordinate axis and the number of cycles on the abscissa axis. The increase in fluorescence is the result of free SYBR Green binding to double-stranded DNA (initially at a very low concentration), which increases roughly 2-fold per amplification cycle until primers and other reagents begin to run out. The fluorescence reaches a maximum at high cycle numbers. A smooth exponential increase in the initial stages of the PCR suggests the amplification of only one product, consistent with the primers being specific. (B) Shows a representative dissociation curve for this reaction, plotting change in fluorescence against change in temperature. As double-stranded DNA product is melted with increasing temperature, it dissociates into two single strands and releases complexed SYBR green. The release of SYBR green results in diminished fluorescence. The central sharp peak seen indicates the homogenous melting behavior of the double-stranded DNA produced by the qRT–PCR, strongly suggesting the production of a single DNA species.
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RESULTS AND DISCUSSION
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Figure 4 describes the process involved in using MultiPriDe.
Figure 4A shows invocation of the script and its request for
GeneID numbers. MultiPriDe then displays the gene's CDS with
a FASTA-formatted header, along with the nucleotide positions
of the splice sites relative to the CDS. As described earlier,
MultiPriDe then makes a directory for each GeneID and places
the Primer3 output files in that folder, as shown in
Figure 4C.
The output files can be read using any text editor; in
Figure 4D
the Emacs terminal utility is used, showing the familiar Primer3
output format.
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Figure 4. The MultiPriDe user experience. (A) The program is invoked using Perl, and asks for GeneID numbers as input. (B) MultiPriDe prints a FASTA-formatted CDS to the screen, along with the locations of splice sites. (C) The directory produced for a given GeneID, containing the primer design files for each splice site. (D) The text file Primer3 output for an individual splice site.
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A comparison between traditional, manual primer design and primer
design conducted using MultiPriDe was undertaken. To manually
design a single pair of primers for a single splice site of
a single gene from our gene set (human cadherin 13, geneID 1012)
we needed 12 min, 45 s to retrieve the genomic sequence, identify
and confirm a single splice site, run Primer3 for that single
location and save all relevant files. The same gene was submitted
to our script; 1 min 26 s was required to identify and develop
100 pairs of primers for each of this gene's 13 splice sites.
This is
7 s per splice site, or roughly 110 times faster than
the manual identification and primer development. MultiPriDe
designed primers for all splice sites of the same 270 genes
in slightly <2 h, running on a 2.0 GHz dual processor Power
Mac G5 (Apple Computer, Cupertino, CA, USA), factory condition
with the exception of 6 GB of RAM, using a 100 Mbit/s internet
connection for retrieving information from NCBI. Of the 270
genes tested, 19 lacked sequence associated with their GeneID,
and therefore primers could not be designed. Experimentally,
our primer selection and optimized qRT–PCR conditions
led to an immediate success rate of 75% among the 251 genes
that we tested experimentally; that is, for each targeted gene,
75% of the first batch of selected primers generated acceptable
qRT–PCR results on the first attempt.
Table 1 describes
the breakdown of first round success for each of the three species
considered. Rachlin and colleagues (
4) describe a 39% chance
for primers designed using their MuPlex utility (which relies
on default parameters for oligonucleotide and salt concentrations
identical to Primer3's default parameters) occurring in more
than 10 genomic locations. Should multiple locations for a given
primer occur across multiple exons, that primer would give confounding
results when used in qRT–PCR. A number of failures are
also due to double peaks that could result from amplification
across an alternative splice site; this has not been quantified.
We feel these phenomena may account for much of our first-round
failures.
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Table 1. Summary of results for qRT–PCR amplifications using the first two selected pairs of MultiPriDe-developed primers
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qRT–PCR primer design for experiments of even modest scope
can quickly become complicated and time-consuming. Manual identification
of ideal target sites is tedious and prone to user-introduced
error; individual queries directed against a public Primer3
server are equally tedious. We do not attempt to quantify error
rate for manual management of sequence and primer design data
in this study. Earlier findings described an error rate of 30–33%
for multiple-step tasks involving text editors, even amongst
highly experienced users (
9). MultiPriDe greatly reduces the
possibility of user-introduced error as all steps short of the
initial GeneID entry are managed entirely by the script. Additionally,
the individual files a user may generate when undertaking these
tasks manually quickly accumulate and may further impact organization.
MultiPriDe was designed with the interests of time and organization
in mind. We are confident that MultiPriDe will be of use to
molecular biologists undertaking qRT–PCR experiments of
any magnitude.
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ACKNOWLEDGEMENTS
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The authors would like to thank Dr J. Boatright and Dr J. Nickerson
for their constructive commentary on this project. The authors
gratefully acknowledge the support of the NSERC, RPB, NIH (NEI)
(P30-EY006360) and the Knights Templar of Georgia. Funding to
pay the Open Access publication charges for this article was
provided by the Knights Templar of Georgia.
Conflict of interest statement. None declared.
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Footnotes
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The authors wish it to be known that, in their opinion, the
first two authors should be regarded as joint First Authors.
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REFERENCES
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- Kuo B, Chen Y, Bohacec S, Johansson O, Wasserman W, Simpson E. SAGE2Splice: unmapped SAGE tags reveal novel splice junctions. PLoS Comput. Biol. (2006) 2:e34.[CrossRef][Medline]
- Li R, Li C. Butyrate induces profound changes in gene expression related to multiple signal pathways in bovine kidney epithelial cells. BMC Genomics (2006) 14:234.
- Qiagen Inc. QuantiTect SYBR Green PCR Handbook. In: Qiagen Inc (2005) Valencia, CA. November 2005 release.
- Rachlin J, Ding C, Cantor C, Kasif S. MuPlex: multi-objective multiplex PCR assay design. Nucleic Acids Res. (2005) 33:W544–W547.[Abstract/Free Full Text]
- Fredslund J, Schauser L, Madsen L, Sandal N, Stougaard J. PriFi: using a multiple alignment of related sequences to find primers for amplification of homologs. Nucleic Acids Res. (2005) 33:W516–W520.[Abstract/Free Full Text]
- Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. In: Bioinformatics Methods and Protocols: Methods in Molecular Biology—Krawetz S, Misener S, eds. (2000) Totowa, NJ: Humana Press. 365–386.
- Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen J.AM. Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res. (2007) 35:W71–W74.[Abstract/Free Full Text]
- Maglott D, Ostell J, Pruitt K, Tatusova T. Entrez Gene: gene-centered information at NCBI. Nucleic Acids Res. (2005) 33:D54–D58.[Abstract/Free Full Text]
- Card S, Moran T, Newell A. The Psychology of Human-Computer Interaction. (1983) Hillsdale, NJ: Erlbaum Associates.
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