1.1 Why did we build qPrimerDB?
Real-time quantitative polymerase chain reaction (qPCR) is a powerful and effective research tool that has been widely used for quantitative detection of gene expression and for identifying differentially expressed genes under various experimental treatments. This time-saving and cost-effective method is frequently used for single nucleotide polymorphism (SNP) genotyping, genetically modified organism (GMO) and pathogen detection, and human in-vitro diagnostics. However, the accuracy and repeatability of qPCR detection are often restrained by primer design and experimental operation factors, leading to considerable variation in detection and incorrect conclusions. It is also noteworthy that the transcription level varies greatly between different gene regions in several eukaryotes investigated (e.g., human, mice, and chicken). To improve the comparability of qPCR detection results obtained by different research groups, only one optimal gene-specific qPCR primer pair should be used to amplify each gene. To ensure the accuracy and comparability of qPCR detection results between different genes, we have developed an automatic gene-specific qPCR primer design and validation workflow based on ePCR and the thermodynamics-based primer specificity checking program MFEprimer-2.0. Until now, we have analyzed 147 genomes and designed 51,091,785 gene-specific qPCR primer pairs, which will be an invaluable resource for qPCR assays.
1.2 What is qPrimerDB?
The qPrimerDB (real-time quantitative PCR Primer Database, https://biodb.swu.edu.cn/qprimerdb) is currently the most comprehensive primer database with a web front end providing gene-specific and pre-computed qPCR primer pairs across 147 important genomes (including human, mice, zebrafish, yeast, thale cress, rice, maize, etc). The qPrimerDB provides an interactive and information-rich web graphical interface to display search and BLAST results as table-based descriptions and associated links. In this database, 3,331,426 best and unique gene-specific qPCR primer pairs were designed for each gene in the 147 genomes sequenced. qPrimerDB also enables batch download of best gene-specific qPCR primer pairs and 51,091,785 possible gene-specific qPCR primer pairs. qPrimerDB thus constitutes a valuable resource for gene expression analysis and will save precious time for researchers.
qPrimerDB is regularly updated. Every update will be reported on the Home page. Visit our site to check if new data or design plans are available.
1.3 How do I cite qPrimerDB?
We hope our database is a useful source of information for researchers, educators, and the interested public. When using these data, please credit qPrimerDB as your source. The current recommended citation format is as follows:
Kun Lu, Tian Li, Jian He, Wei Chang, Rui Zhang, Miao Liu, Mengna Yu, Yonghai Fan, Jinqi Ma, Cunmin Qu, Liezhao Liu, Nannan Li, Ying Liang, Rui Wang, Wei Qian, Zhanglin Tang, Xinfu Xu, Bo Lei, Kai Zhang, Jiana Li. (2017) qPrimerDB: A thermodynamics-based gene-specific qPCR primer database for 147 organisms. Nucleic Acids Res. (accepted).
Permission is not required to use the data for non-profit purposes, although we hope that you will cite qPrimerDB, as described above. If you plan to use the data in qPrimerDB for any for-profit application, please contact us for permission.
2. Database Usage
2.1 What is the data source of qPrimerDB?
So far, qPrimerDB has collected whole genome sequences of 147 important organisms (80 animals, 66 plants, and one fungus), and the corresponding coding/mRNA sequences (Supplementary Table 1). Out of the 147 organisms, genome data of Arabidopsis thaliana was downloaded from The Arabidopsis Information Resource (http://www.arabidopsis.org/), 33 plant genomes were downloaded from Phytozome 12.0 (http://www.phytozome.net), 4 legume genomes were obtained from the Legume Information System (LIS, https://legumeinfo.org), 2 plant genomes were acquired from PLAZA 3.0 (http://bioinformatics.psb.ugent.be/plaza), and the remaining 107 genomes were retrieved from Ensembl (http://www.ensembl.org/).
2.2 Why was MFEprimer-2.0 chosen for evaluating the specificity of qPCR primer?
MFEprimer-2.0 (http://biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0) is a fast thermodynamics-based program for checking the specificity of PCR primers against the background DNA. Unlike traditional sequence alignment-based programs, MFEprimer-2.0 mimics the thermodynamics hybrid process of the primers and the target amplicons and speeds up the binding site searching process using the k-mer algorithm. For more detailed information about MFEprimer-2.0, please read the manual (https://github.com/quwubin/MFEprimer/wiki/Manual) and protocol (http://quwubin.cn/download/QuWB_2015_MFEprimer_Protocol.pdf).
2.3 What is standard to determine the specificity and level?
In this database, all the primers were classified into three levels. The standard for selecting the highest confidence (level 1) gene-specific qPCR primers was primer pair coverage (PPC) = 10. The high confidence (level 2) and low confidence (level 3) gene-specific qPCR primers were selected if the stability of 3′-end of a primer duplex Gibbs free energy (ΔG) is equal or smaller than −9 kcal/mol and −11 kcal/mol, respectively, according to Qu's (Nucleic Acids Res. 40, W205-208, 2012) and Rychlik’s (Biotechniques 18, 84–86, 88–90, 2015) reports.
The PPC of the primer pair is calculated with the following formula:
PPC = Fm × Rm ×（1 - Cvfr）/（Fl × Rl）
where Fm and Rm are sequence overlaps of the forward primer and reverse primer with the template, and Fl and Rl are the lengths of the forward primer and reverse primer, respectively. CVfr is the coefficient of variability of matched length of forward primer and reverse primer. If a pair of primers binds the template completely, the PPC will be 100%. Hence, the primer pairs will be removed from level 1 primer set, if they bind to unintended genes and result in nonspecific amplicons using the PPC cutoff of 10. The remaining primer pairs will be excluded from level 2 and level 3 primer sets if the ΔG of either primer is equal to or smaller than −9 kcal/mol and −11 kcal/mol when binding to mistargeted regions.
2.4 Why are the primers divided into the best and all primers?
GC content is very variable in different genomic regions in many eukaryotes. Recent studies revealed a relatively high correlation between GC content and gene expression in human, mice, and chicken (Arhondakis et al., 2008, 2011; Rao et al., 2013). Hence, it is hard to compare the gene expression detection results using the primer pairs designed from different regions in the same gene..
To avoid the inconsistence in the qPCR detection using different primers, a series of primer pairs at different regions were generated for each gene, and only the best one was included in the best primer set and recommended to users. If a non-specific amplification occurs for a recommended primer pair, researchers can download all primer pairs for target genes, and choose another one for a qPCR assay. We provide the best primer set and all primers for each genome as separate compressed files on the ’Download’, ‘Dataset‘ page.
2.5 How do I browse for information in qPrimerDB?
Please read the manual and follow the example to browse through organisms and identify primer pairs of interest
2.6 How do I search for information in qPrimerDB?
Please read the manual and follow the example of how to search for information of interest by selecting organisms, primer ID, gene ID, or keywords. The bulk search operation is also allowed for users.
2.7 How do I download datasets in qPrimerDB?
The best primer set, all primers, and cDNA or coding sequences for each genome can be downloaded from the ’Download’, ‘Dataset‘ page by choosing an organism and file type (i.e., the best primer, all primers, or sequence).
2.8 What is the meaning of each section in the download datasets?
Both the best primer and all primer files contain 15 columns.
primerID: primer pair ID; GeneID: gene ID; pLevel: level of primer pair; FpID: forward primer ID; RpID: reverse primer ID; Fprimer: forward primer sequence; Rprimer: reverse primer sequence; PPC: primer pair coverage; AmpSize: amplicon size; AmpGC: amplicon GC content; FpTm: melting temperature of forward primer; RpTm: melting temperature of reverse primer; FpDg: Gibbs free energy between the forward primer and its binding site; RpDg: Gibbs free energy between the reverse primer and its binding site; NumExonCorss: number of exon spanned.
2.9 How do I request the primer design for an organism of interest ?
Please fill out the design request form and provide information, including organism, download links for unique cDNA or coding sequence, protein sequence and genome sequence, which are needed for the design request. To increase the running efficiency, links for the gff or gtf file and annotation file are also welcome.
2.10 How specific are the primers in qPrimerDB?
To validate the specificity of amplification and to check for the presence of non-specific amplicons, we performed qPCR assays for 66 randomly selected genes in thale cress (Arabidopsis thaliana), oilseed rape (Brassica napus), rice (Oryza sativa), sweet orange (Citrus sinensis), silkworm (Bombyx mori), and zebrafish (Danio rerio). In the qPCR experiments, only the best primer pair for each gene from qPrimerDB was selected. To ensure the reliability of experimental results, the setup and validation of qPCR were performed under conditions prescribed in the MIQE guidelines. Based on melting curve and gel electrophoresis, all the selected primer pairs were shown to be highly specific with a high amplification efficiency.
2.11 Questions not answered in this FAQ ?
If you have any inquiries that are not addressed in the FAQs, feel free to contact us via email or the ’Contact Us‘ feedback form.
Comments & Collaborations
We are very interested in receiving your comments, suggestions for improvement, and guidance. Please contact us via e-mail or use the online feedback form on the 'Contact Us' tab from the ‘Help’ page. We would like to invite the scientific community to submit their qPCR detection results. This information will be very helpful to the community.
We also encourage the scientific community to submit their qPCR primer development request for your own genome or transcriptome. We will continuously update our database and add more species in the qPrimerDB.