PrimerBank: a PCR primer database for quantitative gene expression analysis, 2012 update.

Optimization of primer sequences for polymerase chain reaction (PCR) and quantitative PCR (qPCR) and reaction conditions remains an experimental challenge. We have developed a resource, PrimerBank, which contains primers that can be used for PCR and qPCR under stringent and allele-invariant amplification conditions. A distinguishing feature of PrimerBank is the experimental validation of primer pairs covering most known mouse genes. Here, we describe a major update of PrimerBank that includes the design of new primers covering 17,076 and 18,086 genes for the human and mouse species, respectively. As a result of this update, PrimerBank contains 497,156 primers (an increase of 62% from the previous version) that cover 36,928 human and mouse genes, corresponding to around 94% of all known protein-coding gene sequences. An updated algorithm based on our previous approach was used to design new primers using current genomic information available from the National Center for Biotechnology Information (NCBI). PrimerBank primers work under uniform PCR conditions, and can be used for high-throughput or genome-wide qPCR. Because of their broader linear dynamic range and greater sensitivity, qPCR approaches are used to reanalyze changes in expression suggested by exploratory technologies such as microarrays and RNA-Seq. The primers and all experimental validation data can be freely accessed from the PrimerBank website, http://pga.mgh.harvard.edu/primerbank/.

PrimerBank: a resource of human and mouse PCR primer pairs for gene expression detection and quantification.

PrimerBank (http://pga.mgh.harvard.edu/primerbank/) is a public resource for the retrieval of human and mouse primer pairs for gene expression analysis by PCR and Quantitative PCR (QPCR). A total of 306,800 primers covering most known human and mouse genes can be accessed from the PrimerBank database, together with information on these primers such as T(m), location on the transcript and amplicon size. For each gene, at least one primer pair has been designed and in many cases alternative primer pairs exist. Primers have been designed to work under the same PCR conditions, thus facilitating high-throughput QPCR. There are several ways to search for primers for the gene(s) of interest, such as by: GenBank accession number, NCBI protein accession number, NCBI gene ID, PrimerBank ID, NCBI gene symbol or gene description (keyword). In all, 26,855 primer pairs covering most known mouse genes have been experimentally validated by QPCR, agarose gel analysis, sequencing and BLAST, and all validation data can be freely accessed from the PrimerBank web site.

A comprehensive collection of experimentally validated primers for Polymerase Chain Reaction quantitation of murine transcript abundance.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Sub-proteome differential display: single gel comparison by 2D electrophoresis and mass spectrometry.

Two-dimensional (2D) gel electrophoresis and mass spectrometry (MS) have been used in comparative proteomics but inherent problems of the 2D electrophoresis technique lead to difficulties when comparing two samples. We describe a method (sub-proteome differential display) for comparing the proteins from two sources simultaneously. Proteins from one source are mixed with radiolabelled proteins from a second source in a ratio of 100:1. These combined proteomes are fractionated simultaneously using column chromatographic methods, followed by analysis of the pre-fractionated proteomes (designated sub-proteomes) using 2D gel electrophoresis. Silver staining and (35)S autoradiography of a single gel allows precise discrimination between members of each sub-proteome, using commonly available computer software. This is followed by MS identification of individual proteins. We have demonstrated the utility of the technology by identifying the product of a transfected gene and several proteins expressed differentially between two renal carcinoma proteomes. The procedure has the capacity to enrich proteins prior to 2D electrophoresis and provides a simple, inexpensive approach to compare proteomes. The single gel approach eliminates differences that might arise if separate proteome fractionations or 2D gels are employed.