A comprehensive collection of experimentally validated primers for Polymerase Chain Reaction quantitation of murine transcript abundance
© Spandidos et al; licensee BioMed Central Ltd. 2008
Received: 19 September 2008
Accepted: 24 December 2008
Published: 24 December 2008
Quantitative polymerase chain reaction (QPCR) is a widely applied analytical method for the accurate determination of transcript abundance. Primers for QPCR have been designed on a genomic scale but non-specific amplification of non-target genes has frequently been a problem. Although several online databases have been created for the storage and retrieval of experimentally validated primers, only a few thousand primer pairs are currently present in existing databases and the primers are not designed for use under a common PCR thermal profile.
We previously reported the implementation of an algorithm to predict PCR primers for most known human and mouse genes. We now report the use of that resource to identify 17483 pairs of primers that have been experimentally verified to amplify unique sequences corresponding to distinct murine transcripts. The primer pairs have been validated by gel electrophoresis, DNA sequence analysis and thermal denaturation profile. In addition to the validation studies, we have determined the uniformity of amplification using the primers and the technical reproducibility of the QPCR reaction using the popular and inexpensive SYBR Green I detection method.
We have identified an experimentally validated collection of murine primer pairs for PCR and QPCR which can be used under a common PCR thermal profile, allowing the evaluation of transcript abundance of a large number of genes in parallel. This feature is increasingly attractive for confirming and/or making more precise data trends observed from experiments performed with DNA microarrays.
Quantitative polymerase chain reaction (QPCR) has become a widely applied technique for quantitative gene expression analysis [1, 2]. The technique is frequently used to validate and improve the precision of measurement of differences in transcript abundance detected by DNA microarray experiments . In QPCR, product formation is monitored at the end of each thermal cycle by determining the strength of a fluorescent signal that is proportional to the amount of product [4, 5]; QPCR thus provides more information than can be inferred from signal detected at the end of multiple cycles of reaction, as in conventional PCR analysis [6–8]. Because data can be collected from the exponential phase of the reaction a generally reliable quantitation of target DNA concentration can be achieved . Detection of QPCR product concentration is usually accomplished by one of two general fluorescence-based approaches: the measurement of a target sequence-selective signal arising from a conformational change in a labeled primer, or the measurement of total DNA formed during the reaction. In the former method, target-specific probes containing fluorophores, such as hydrolysis probes [10–13], dual hybridization probes , molecular beacons  or scorpions [16, 17] are designed. These detection systems provide partial protection against the risk of generation of signals from off-target amplicons but the primers are considerably more expensive to generate than conventional unlabeled primers. In a more widely practiced variant of QPCR, sequence non-selective fluorescent dyes that bind to double-stranded DNA, such as SYBR Green I, are used [18, 19]. The quantum yield of SYBR Green I dye intercalated into double-stranded DNA is much greater than the quantum yield of free dye, leading to an increase in fluorescence intensity that, at saturating dye concentration, is proportional to DNA concentration . This yields a simple inexpensive way to measure product amplicon formation. However, the contribution of fluorescence from DNA arising by amplification of undesired sequences cannot be determined without some additional measure, such as thermal dissociation analysis .
Several online resources have been described that can be used to design primers for PCR and QPCR [22–25] and are useful for gene expression analysis, when a small number of genes are of interest. We have previously described a resource of designed primers that can be used for real-time PCR with sequence independent detection methods, such as SYBR Green I detection, and that can work under a common PCR thermal profile . Amplification of undesired sequences is a common problem in QPCR, and poses greater difficulties when the amplification conditions cannot be tailored to the primer pair of interest, as for example would be the case for massively parallel QPCR. The primer design algorithm used for the selection of primers for this study was based on a previous approach to the prediction of oligonucleotides for the study of protein coding regions by microarrays , but differed by the addition of filters thought to be important for PCR primer specificity. Primers were designed from cDNA sequence information and the principal filter for cross-reactivity was the rejection of primers containing contiguous residues (15 bases or longer) present in other sequences . Additionally, the selected primer pairs had no self-complementarity, low 3' end stability and high complexity. Low complexity regions may contribute to primer cross-reactivity , so they were excluded using the DUST program . The primer Tms were in the same range, as well as their GC contents. Short amplicons (60–350 bp) were favored during primer selection, but in some cases 100–800 bp amplicons were also considered when the design criteria could not be met for shorter amplicons.
Statistics of primers contained in the PrimerBank database.
PrimerBank primer design and gene representation.
Mouse primer pair design
Number of mouse primer pairs or genes
Primer pairs with no redundancy
Primer pairs with 2 target genes
Primer pairs with more than 2 target genes
Total number of primer pairs
Total number of genes represented
Total number of genes not represented
High-throughput primer validation procedure
Classification of failed PrimerBank primer pairs.
Type of failure
Reasons for failure
Number of primer pairs
% from total analyzed
No amplification detected
Agarose gel failures:
No band observed on gel
Multiple bands observed on gel
Wrong size band observed on gel
Faint band observed on gel
Failed based on gel analysis criteria (G1–G4)
Low sequence quality
Sequences obtained did not match to the expected sequences
Low match length between sequences obtained and the expected sequences
Low % identity between sequences, expected sequences were not 1st matches
Failed based on BLAST analysis criteria (B1–B3)
A few representative examples of primer pairs are described [see Additional files 1, 2, 3, 4, 5], to demonstrate in detail the analysis of the results generated from the high-throughput primer validation procedure. Data are shown for five successful primer pairs, five primer pairs that failed based on agarose gel electrophoresis analysis and five primer pairs that failed based on BLAST analysis. Information on these primer pairs, such as PrimerBank IDs, primer sequences and amplicon lengths, is shown here [see Additional file 4]. More information on these primers, such as their Tm and location on the gene, can be found in PrimerBank, as well as alternative primer pairs designed for these transcripts.
PrimerBank user interface
All data generated from the high-throughput primer validation procedure can be freely accessed from PrimerBank http://pga.mgh.harvard.edu/primerbank/. See Figure 1 for the PrimerBank homepage. Users can search the PrimerBank database for primers for their gene of interest using several search terms such as: GenBank accession number, NCBI protein accession number, NCBI gene ID, PrimerBank ID, NCBI gene symbol or gene description (keyword). Search results include primer sequences together with some information about the primers, such as expected amplicon size and Tm. cDNA and amplicon sequences, and validation data can be viewed by clicking on the appropriate links. All validation data can be accessed from PrimerBank, since the validation criteria may be different from the criteria of the users. Also, users can use a BLAST tool found on the PrimerBank homepage (see Figure 1), to find any primers contained in the PrimerBank database that would amplify their sequence of interest. A BLAST tool for the PCR product sequence obtained from the validation procedure can be used to query the NCBI database and this can be found on the validation data webpage. The QPCR and reverse transcription protocols can be found on PrimerBank, as well as a troubleshooting guide.
Analysis of failed primer pairs
From the total agarose gel failed reactions, 46.7% were due to multiple amplification products apparent by gel electrophoresis. 13.8% of the total failed reactions were due to undesired amplification, seen as the wrong size band on the gel. 4.8% of the total failed reactions were due to poor amplification, and 34.7% of the total failed reactions were due to no amplification taking place. Multiple or undesired amplifications accounted for the majority (60.5%) of the agarose gel failed reactions. These may represent undocumented transcripts or splice isoforms that could have been amplified in addition to or instead of the expected transcripts. For the reactions that failed because no amplification had taken place, the template sequences may not have been present or present in very low copy number.
Validation of primer pairs that failed amplification using genomic DNA
From the high-throughput PrimerBank mouse primer pair validation, 1745 samples (6.5%) failed because of no amplification, as seen from the QPCR amplification plots. From the gene description information we found several to belong to olfactory receptors, vomeronasal receptors, transcription factors and low abundance transcripts while others were of unknown function or RIKEN sequences (data not shown). In order to investigate the possibility that the templates for the failed amplification primer pairs were not expressed in the cDNA sample used, we repeated these reactions using genomic DNA as a template. It can be difficult to achieve amplification using genomic DNA as template in general, due to its complexity. However, it can be used successfully if technical difficulties are overcome and can be useful as a universal template as it contains a copy of all genes, and the same amount of template is present for all single-copy genes . We have found that enzymatic digestion (such as Eco RI/Bam HI digestion used here) can be used for reduction of the complexity of the DNA and thus higher amplification rates. We matched 864 primer pairs to mouse genome sequences obtained from the UCSC genome browser. The remainder of the sequences could not be matched, probably because they were located on exon junctions. 640 of these primer pairs have no Eco RI/Bam HI restriction sites in their expected PCR amplicons, and were used with Eco RI/Bam HI digested DNA template to prepare the validation reactions. We tested 192 representative samples, from the 1745 total number of failed primer pair samples, whose expected PCR amplicon lengths range from 60 bp to 123 bp and whose amplicons have no Eco RI/Bam HI restriction sites. 50 ng Eco RI/Bam HI digested 129 mouse ES cell genomic DNA was used per 25 μl PCR reaction.
The amplification plots of all 192 samples (2 × 96 well plates) are shown here [see Additional files 6, 7]. The success rate of QPCR based on the amplification plots was high: 88.5% for the first plate [see Additional file 6] and 90.6% for the second plate [see Additional file 7]. However, Ct values differed significantly, from roughly 23 to 40 [see Additional files 6, 7]. The location of the reactions on the plate did not explain this variation. The samples were also analyzed by agarose gel electrophoresis and sequenced (data not shown). Sequences obtained were BLAST analyzed and matched to the expected sequences, confirming that the correct templates had been amplified (data not shown). Therefore, these primer pairs had originally failed because their respective templates were not present in the cDNA sample used and not because of poor primer design, in general.
Uniformity of amplification and technical replicate tests
We next set out to determine the uniformity of amplification using fully validated PrimerBank primer pairs ie. primer pairs that had been successful in all steps of the validation procedure. 96 primer pairs were chosen with expected PCR amplicon length ranging from 80 bp to 120 bp and containing no Eco RI/Bam HI restriction sites in their sequences. Both forward and reverse primers were chosen to be on the same exon in order to amplify the same template on genomic DNA. Eco RI/Bam HI digested 129 mouse ES cell genomic DNA was used as template. After digestion the DNA was purified for PCR by phenol extraction and ethanol/salt precipitation. 50 ng of DNA template was used per 25 μl PCR reaction, which was found by optimization experiments to give a reasonable Ct value.
In order to determine the technical reproducibility of the QPCRs, five 96 well plate assays were prepared using the same technical procedure. Reactions were set up using the same 96 primer pairs and DNA template (129 mouse ES cell Eco RI/Bam HI digested genomic DNA) that were used for the uniformity of amplification test. The coefficients of variation for each 96 well plate assay are all < 0.1 and the average coefficient of variation for all assays is 0.07 [see Additional file 8]. The individual primer pair Cts for each 96 well plate assay and coefficients of variation are shown here [see Additional file 9]. Ct data from each assay initially did not pass the statistical normality test. The Ct values were normalized, using the formula:
(LnCt - LnCtav)/SD,
where LnCt is the natural logarithm of the Ct value used, LnCtav is the natural logarithm of the average Ct value of the assay and SD is the standard deviation of the LnCt values for each assay, and outliers were removed. The normalized data passed the normality test, so the data appear to be log normal. The plots of the frequency distributions of the log normal data are shown here [see Additional file 10].
Analysis of pipetting variation during liquid transfer of the fluid handling system was carried out and the transfer efficiency of the robot was found to be 97.3% [see Additional file 11]. The data from the liquid transfer test passed the statistical normality test only after the 9 lowest value outliers were removed (data not shown), but the coefficients of variation are low (less than 0.03) [see Additional file 11]. Variation in liquid transfer can only account for a small amount of the variation observed in QPCR reactions, and hence other factors must be responsible for the differences observed in Ct values.
SYBR Green I sequence specificity
The SYBR Green I dye has been widely used as a non-sequence specific dye for fluorescence detection of QPCR products . Studies of SYBR Green I-DNA binding showing some sequence specificity of the dye have been reported but these have not been conclusive [20, 33, 34]. We investigated whether SYBR Green I is sequence specific by adding the dye to a series of amplicons and taking fluorescence readings. 8 amplicons of increasing length and 7 amplicons of increasing AT% [see Additional file 12] were used, whose concentrations were accurately determined (see methods). From these experiments, we did not observe any length dependent or AT/GC dependent sequence specificity of SYBR Green I [see Additional file 13]. However, we cannot exclude the possibility that SYBR Green I can show specificity to sequences such as homopolymer regions of DNA  or specific sequences. We also investigated whether SYBR Green I dye binding is sequence specific by estimating the number of PCR product molecules at threshold using the ABI PRISM 7000 Sequence Detection System (Applied Biosystems) [35, 36]. For this, the same 14 amplicons as above were used and a template titration series of reactions was prepared for each amplicon. SYBR Green I threshold cycle (Ct) fluorescence will be the same for all amplicons (and all reactions), since the same threshold was used to compare all reactions. However, if SYBR Green I is sequence specific, this fluorescence will correspond to a different number of molecules at threshold for each amplicon. These experiments were inconclusive, as the stochastic error was too large to be able to accurately determine the molecules detected at the threshold (data not shown).
Estimation of QPCR amplification efficiency
The most common method for the calculation of the amplification efficiency of a QPCR reaction requires preparation of a series of serial dilutions of the sample and creation of a standard curve, whereby efficiency is estimated from the slope of the standard curve [36, 37]. However, this method does not provide an accurate value of the efficiency, as the efficiency can vary between different reactions and as input concentration changes. A number of analytical methods have been described for the calculation of the amplification efficiency of a reaction from single reaction kinetics  (for a correction in equation 3 of this paper see: ), [40–42]. These methods can be more accurate and, when automated, less laborious compared to the standard curve method . Using the following analytical method, we estimated the amplification efficiency values for 13 QPCRs using PrimerBank primer pairs that had been previously used. The log2 fluorescence data was plotted versus the Ct number and the slope of the linear regression was taken to be equal to the efficiency of each reaction [see Additional file 14]. Cycle values closest to the Ct were used, as this region will be the most accurate. The efficiency values ranged from 79% to 96% [see Additional file 14]. Replicates can be used to improve accuracy when using either the standard curve or analytical single reaction kinetics methods [39, 44].
We compared amplification efficiency estimation using the standard curve and analytical methods in order to determine the accuracy of each method using the same 13 PrimerBank primer pairs as above [see Additional file 15]. Either the log2 of pg of input template DNA data, for the standard curve method, or the log2 fluorescence data, for the analytical method, was plotted versus the Ct number. Ct was the independent variable and log2 of pg of input template DNA/fluorescence was the dependent variable. The slope of the linear regression was taken to be equal to the efficiency of each reaction. From these results the analytical method shows a smaller variance of efficiency values and the range is smaller compared to the standard curve method [see Additional file 15]. One-way ANOVA analysis was done to determine if amplification efficiency varied significantly between different PrimerBank primer pairs, using each primer pair in a series of titration reactions of template DNA [see Additional file 16]. The average efficiency, standard deviation and coefficient of variation for each group of primer pairs are shown here [see Additional file 17]. The P value is > 0.05 (0.7338) therefore the amplification efficiency is similar between these groups.
Log2pgDNA = β0 + βCtxCt + ε,
where Log2pgDNA is the dependent variable, β0 is the intercept, βCt is the regression coefficient for the x independent variable, and ε is the error. Equation 1 can be used for the standard curve method. Log2Fluorescence = β0 + βxxc + ε,
where Log2Fluorescence is the dependent variable, β0 is the intercept, βx is the regression coefficient for the x independent variable of cycle c, and ε is the error. If βx = 1, amplification efficiency is 100%. Equation 2 can be used for the analytical methods.
PrimerBank primer pair gene location
Primer pair location with respect to exons.
F primer exon location
R primer exon location
Source of DNA template
A commercial composite mouse RNA preparation was chosen as the source of DNA template for QPCRs, which contains RNA from a panel of eleven different mouse cell types for a good representation of the majority of mouse genes. The composite mouse RNA is composed of total RNA from: whole embryo, embryonic fibroblasts, kidney, liver, lung, B-lymphocyte, T-lymphocyte, mammary gland, muscle, skin and testis. The success rate of the high-throughput PrimerBank primer validation experiments was high as seen both from agarose gel and BLAST analysis. We validated some of the failed reactions using genomic DNA as template, and found that most of the failures in which no PCR product had formed could be due to very little or no cDNA present in the source of DNA template. In order to increase amplification success, specific tissues may be used as sources of cDNA templates where expression of the genes of interest is known.
The PrimerBank primer design was based on a successful approach for the prediction of oligonucleotides for the interrogation of protein coding regions by microarrays . However the primer design differs by the addition of filters that are thought to be important for primer specificity . All primers have been designed to work using a relatively high annealing temperature of 60°C and this temperature was used throughout the primer validation experiments described here. High annealing temperatures help reduce non-specific amplification. A high percentage of the total failed samples were due to undesired or multiple amplification, however this may have been for other reasons such as new unidentified genes or splice isoforms. In 3.9% of the cases where multiple bands could be seen on the agarose gel and in 14.6% of the cases where bands of other than the expected size could be seen on the agarose gel, no sequencing information was obtained. Also, 29.7% and 55.2% respectively, did not match to the expected sequences by BLAST. So, sequence homology existed in most cases of undesired or multiple amplification. From the genome-wide primer validation experiments presented here, we have found a high success rate of primer pairs that amplify the transcripts for which they had been designed. For primer pairs that failed because no amplification could be detected, we found that the reason for which they had initially failed was because their target sequences were not present in the target cDNA used. Another reason for failure in the high-throughput validation procedure, may be that protein coding genes in the human genome are fewer than previously thought, and the same may apply to the mouse genome .
A collection of potential new splice isoforms
As mentioned previously, larger than expected or multiple bands were visible on the agarose gel for some samples, however, sequences for these matched confidently by BLAST to the expected sequences. Therefore, the template sequences amplified in these cases could be new genes or splice isoforms. These unrecognized genes or splice isoforms may contribute to primer cross reactivity which results in a lower success rate on the agarose gels. Good primer design depends on accurate genomic information about genes and splice isoforms and it is suggested that many unidentified genes and splice isoforms could exist. All primer pairs that failed because of non-specific amplification, but when BLAST analyzed matched to the expected sequence, could have amplified new non-identified isoforms. This information would be very useful for other researchers, in addition to other strategies for identifying new genes and splice isoforms [51, 52]. PrimerBank primers could also be used for determining copy-number variation of a gene or splice isoform [53, 54].
The PrimerBank database
Several online databases exist containing experimentally validated primers, however, only a few thousand primer pairs are currently present in these databases [55–57]. We have previously designed PCR primers for the human and mouse genomes, which are available from PrimerBank . The PrimerBank database currently contains 306800 primers for the mouse and human genomes and is tightly integrated with information from the NCBI databases. PrimerBank has been designed so that researchers can search for primers for their gene of interest using several search terms such as: GenBank accession number, NCBI protein accession number, NCBI gene ID, PrimerBank ID, NCBI gene symbol or gene description (keyword). Currently, all validated primers can be retrieved by searching PrimerBank. In many cases, alternative primer pairs for genes also exist in PrimerBank. NCBI sequences have been attached to the primer information page and NCBI LocusLink indices have been used internally for gene locus mapping. All primers have uniform properties such as Tm, length and GC content and can work using the same PCR conditions.
We tested by QPCR 26855 PrimerBank mouse primer pairs in order to determine if they can successfully amplify the genes for which they had been designed. We identified 17483 primer pairs that amplify unique sequences that correspond to distinct murine transcripts. All primers have been used under a common PCR thermal profile, allowing the experimentally validated primer collection to be used to evaluate the transcript abundance of a large number of genes in parallel. We used genomic DNA as a template to validate primer pairs that had initially failed by QPCR and provided explanations for the various modes of failure. We determined the uniformity of amplification of the QPCRs using 96 PrimerBank primer pairs. From the uniformity experiments, we found a small variation in Cts which could be due to differences in PCR product length and/or stochastic variation. However, overall amplification appears to be uniform using PrimerBank primers. We investigated the reproducibility of the QPCRs, using the same 96 primer pairs that were used for the uniformity experiments, by comparing Ct values between five technical replicate plates and found coefficients of variation to be low. In addition, SYBR Green I sequence specificity was investigated, using a set of sequences differing in length and base composition. We found no SYBR Green I specificity for the sequences used, but cannot exclude SYBR Green I specificity towards specific sequence motifs. Furthermore, we calculated the efficiency of the reactions from single reaction kinetics data and found the estimated efficiencies to be within a reasonable range, and also that the efficiency can vary between different templates. PrimerBank provides a useful tool for quantitative gene expression analysis by QPCR and facilitates high-throughput studies.
High-throughput primer validation procedure
Oligonucleotides for QPCR were synthesized at Synthesis Core lab of Center for Computational and Integrative Biology at Massachusetts General Hospital. The quality and quantity of the synthesized oligonucleotides were determined by capillary elecrophoresis using the MCE 2000 (CombiSep) instrument and by OD260 reading using the Spectra Max Plus Spectrophotometer (Molecular Devices). Forward and reverse primer mixtures were normalized to 2 μM of each primer for use in QPCR.
Preparation of cDNA sample
Universal Mouse Reference total RNA (Stratagene) was used for the preparation of the cDNA sample. Reverse transcription using random hexamers was performed using the Superscript First-Strand Synthesis System for RT-PCR (Invitrogen). Based on the recommended protocol, 20 μg of total RNA was used for each reaction and cDNA samples prepared were in a final volume of 84 μl. The quality of the individual first strand cDNA preparations was tested in a QPCR reaction using mouse actin primers (PrimerBank ID: 6671509a1, 6671509a1F: GGCTGTATTCCCCTCCATCG, 6671509a1R: CCAGTTGGTAACAATGCCATGT).
QPCRs were performed in polypropylene 96 well plates on the ABI PRISM 7000 Sequence Detection System and ABI 7300 Real-Time PCR System (both from Applied Biosystems). SYBR Green PCR Master mix (Applied Biosystems) or Absolute Q-PCR SYBR Green ROX mix (ABgene) were used. For each reaction, 12.5 μl of the 2× SYBR Green PCR mix were added to 2.5 μl of 2 μM forward and reverse primer mix (final concentration of each primer is 200 nM), 1 μl of cDNA and made to 25 μl with water. The Biomek FX Laboratory Automation Workstation (Beckman Coulter), as well as manual pipetting, was used to prepare the reactions. PCR conditions used were the following: 50°C for 2 minutes (step 1), 95°C for 10 minutes (for Applied Biosystems PCR mix) or for 15 minutes (for ABgene PCR mix) (step 2), 95°C for 15 seconds, 60°C for 30 seconds, 72°C for 30 seconds (step 3 – repeated another 39 times ie. 40 cycles in total). In some QPCRs an additional elongation step was added at 72°C for 10 minutes (step 4). Dissociation curves were obtained by heating and cooling the samples at: 95°C for 15 seconds, 60°C for 30 seconds, 95°C for 15 seconds. DNA was renatured for agarose gel electrophoresis using the following conditions: 50°C for 2 minutes (step 1), 95°C for 15 seconds, 60°C for 30 seconds (step 2 – repeated one more time) and 72°C for 5 minutes (ABI PRISM 7000 Sequence Detection System) or 95°C for 30 seconds, 60°C for 2 minutes (ABI 7300 Real-Time PCR System).
Preparation of samples for agarose gel electrophoresis and sequencing
PCR products were purified using Standard Performa 96 well plates and QuickStep 2 SOPE resin (both from EDGE BioSystems), following the recommended procedure.
Agarose gel electrophoresis of purified QPCR products
For each sample 10 μl of 2× Orange G loading buffer (composition shown below) was added to 5 μl of the purified PCR product and made to 20 μl with water. Samples were prepared in 96 well plates using the Biomek FX Laboratory Automation Workstation (Beckman Coulter) and using the same instrument applied to 2% agarose 96 well E-gels (Invitrogen). For 10× Orange G loading buffer, a solution of 30% Ficoll 400 (AlfaAesar), 10 mM EDTA (Sigma) was prepared and Orange G dye (Fisher Scientific) was added for color. E-Gel Low Range Quantitative DNA Ladder (Invitrogen) was used as a marker for PCR product size. The gels were run for 12 minutes on the E-Gel 96 Base (Invitrogen) and analyzed using the E-Editor Software (Invitrogen).
Sequencing of purified QPCR products
Purified QPCR products were sequenced at Sequencing Core lab of Center for Computational and Integrative Biology at Massachusetts General Hospital.
NCBI BLAST analysis
Sequences obtained were BLAST analyzed as batch sets against the NCBI database . In order to identify successful samples, the main parameters considered were the alignment length, the expected sequence match position to the sequence returned by NCBI BLASTn and the percent identity of the two sequences. If more than 50% of the length of the expected PCR product sequence aligned with the expected sequence as first match and there was more than 92% identity between the sequences, this was considered to be a successful sample. In cases where a primer pair had been designed to also amplify a redundant gene and the redundant gene matched first to the sample, the reaction was still considered successful. In these cases the primers have been designed to amplify the same region of the two sequences, so it is not possible to determine by agarose gel or BLAST analysis if one or the other species was amplified during PCR.
Preparation of digested genomic DNA for QPCR
129 Embryonic Stem cell mouse genomic DNA (isolated by ethanol precipitation) was used. The DNA was digested completely using Eco RI and Bam HI restriction enzymes. Digests were made by adding 20 μl Eco RI buffer (10×) (New England Biolabs), 20 μl 10× BSA, 4 μg DNA, 40 U Bam HI (New England Biolabs), 40 U Eco RI (New England Biolabs) and water to 200 μl total volume. Digests were incubated at 37°C for 4 hours and 30 minutes and heat inactivated at 75°C for 10 minutes. The digested DNA was phenol extracted and ethanol/salt precipitated. DNA pellets were resuspended in TE pH 8.0.
QPCRs for uniformity, technical replicate and primer validation tests
QPCRs were performed in polypropylene 96 well plates on the ABI 7300 Real-Time PCR System (Applied Biosystems). For each reaction, 12.5 μl of Absolute Q-PCR SYBR Green ROX mix (ABgene) were added to 2.5 μl of 2 μM forward and reverse primer mix (final concentration of each primer is 200 nM), 1 μl of 50 ng/μl Bam HI/Eco RI digested genomic DNA and made to 25 μl with water. The following PCR conditions were used: 50°C for 2 minutes (step 1), 95°C for 15 minutes (step 2), 95°C for 15 seconds, 60°C for 30 seconds, 72°C for 30 seconds (step 3 – repeated another 39 times ie. 40 cycles in total), 72°C for 10 minutes (step 4). Dissociation curves were obtained by heating and cooling the samples at: 95°C for 15 seconds, 60°C for 30 seconds, 95°C for 15 seconds.
Large-scale amplicon preparation for SYBR Green I sequence specificity experiments
Amplicons were prepared large-scale by PCR, in two steps. For the first step PCR, 75 μl PCR reactions were prepared for each sample. For each reaction, 37.5 μl Absolute Q-PCR SYBR Green ROX mix (ABgene) were added to 3 μl of 5 μM primer pair mix (final concentration of each primer is 200 nM), 3 μl universal mouse cDNA (see: 'preparation of cDNA sample' section in methods) and made up to 75 μl with water. The following PCR conditions were used: 95°C for 15 minutes (step1), 95°C for 15 seconds, 60°C for 30 seconds, 72°C for 30 seconds (step 2 – repeated another 39 times ie. 40 cycles in total), 72°C for 10 minutes (step 3). The PCR products were purified using the MinElute PCR purification kit (Qiagen). Purified amplicons were used as templates in large-scale 40× 100 μl PCRs, each reaction containing 50 μl 2× LC1v3 buffer (40 mM Tris-HCl pH8.8, 40 mM KCl, 40 mM ammonium sulfate, 4 mM MgCl2, 200 μg/ml BSA, 0.2% Triton X-100, 400 μM dNTP mix, 2.5 M betaine), 4 μl of 5 μM forward and reverse primer mix, DNA template, 1 μl Taq polymerase and water to 100 μl. The PCR conditions used were the following: 95°C for 3 minutes (step 1), 95°C for 15 seconds, 60°C for 30 seconds, 72°C for 30 seconds (step 2 – repeated another 39 times ie. 40 cycles in total), 72°C for 10 minutes (step 3).
PCR reactions were phenol extracted and isopropanol precipitated. DNA pellets were resuspended in TE pH8.0. DNA was purified using Performa DTR Gel Filtration Cartridges (EDGE BioSystems), following the recommended procedure. Amplicon concentrations were determined by taking OD260 readings of each preparation using the ND-1000 Spectrophotometer (Nanodrop). The average value was taken and the OD260 reading from a no DNA template control was subtracted, in order to remove the contribution from primers and buffer components to the spectrophotometric absorption.
SYBR Green I sequence specificity experiments
DNA samples in 1× Absolute Q-PCR SYBR Green ROX mix (ABgene) were pipetted into OptiPlate-96F black 96 well plates (Perkin Elmer). SYBR Green I fluorescence was detected using the Analyst AD fluorescence plate reader (Molecular Devices) by excitation at 485 nm and emission at 530 nm (505 nm dichroic mirror).
Robotic and manual liquid transfer test
5 μl of 10 mM dNTP solution were added to 95 μl water and the OD260 readings were taken using the Spectra Max Plus Spectrophotometer (Molecular Devices).
Primer genome location analysis
Mouse genome sequences were downloaded from the UCSC genome browser  and the primer pair sequences were matched by BLASTn to the genome sequences, to identify the primer locations with respect to exons.
We thank our colleagues Chen Liu and Don Dwoske for their technical help, the Automation, Synthesis and Sequencing Core labs of Center for Computational and Integrative Biology at Massachusetts General Hospital for their contributions and Naifang Lu for the 129 ES cell mouse genomic DNA. This work was supported by the National Institutes of Health Program for Genomic Applications, grant U01 HL66678.
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