Double-check the zebrafish 18s rRNA qPCR primers: they may be wrong

A widely used qPCR primer for zebrafish gene rna18s (18s rRNA, or 18s), with the sequence of 5'-TCGCtaGTtGGCATCGTTTAtG-3', is found to be incorrect. Initially designed for rainbow trout (Oncorhynchus mykiss) rna18s, the primer has four different nucleotides from the zebrafish sequence 5'-TCGCGGGTCGGCATCGTTTACG-3' (indicated in bold/underlined, lowercase letters for rainbow trout and uppercase letters for zebrafish). Since its first use in zebrafish in 2006, this mismatched primer has been clearly stated to be used in at least 50 publications and may have affected hundreds or more in publications citing them. For a sensitive, quantitative method as qPCR, this error must be corrected as soon as possible in the zebrafish community by using rna18s primer sets with accurate sequences, such as those summarized and newly designed by this article.

MetaFunPrimer: primer design for targeting genes observed in metagenomes

ABSTRACTHigh throughput primer design is needed to simultaneously design primers for multiple genes of interest, such as a group of functional genes. We have developed MetaFunPrimer, a bioinformatic pipeline to design primer targets for genes of interests, with a prioritization based on ranking the presence of gene targets in references, such as metagenomes. MetaFunPrimer takes inputs of protein and nucleotide sequences for gene targets of interest accompanied by a set of reference metagenomes or genomes for determining genes of interest. Its output is a set of primers that may be used to amplify genes of interest. To demonstrate the usage and benefits of MetaFunPrimer, a total of 78 HT-qPCR primer pairs were designed to target observed ammonia monooxygenase subunit A (amoA) genes of ammonia-oxidizing bacteria (AOB) in 1,550 soil metagenomes. We demonstrate that these primers can significantly improve targeting of amoA-AOB genes in soil metagenomes compared to previously published primers.IMPORTANCEAmplification-based gene characterization allows for sensitive and specific quantification of functional genes. Often, there is a large diversity of genes represented for a function of interest, and multiple primers may be necessary to target associated genes. Current primer design tools are limited to designing primers for only a few genes of interest. MetaFunPrimer allows for high throughput primer design for functional genes of interest and also allows for ranking gene targets by their presence and abundance in environmental datasets. This tool enables high throughput qPCR approaches for characterizing functional genes.

The Quantification of Antibody Elements and Receptors subunit expression using qPCR: The Design of VH, VL, CH, CL, FcR subunits primers for a more holistic view of the immune system

The expression levels of Immunoglobulin elements and their receptors are important markers for health and disease. Within the immunoglobulin locus, the constant regions and the variable region families are associated with certain pathologies, yet a holistic view of the interaction between the expression of the multiple genes remain to be fully characterized. There is thus an important need to quantify antibody elements, their receptors and the receptor subunits in blood (PBMC cDNA) for both screening and detailed studies of such associations. Leveraging on qPCR, we designed primers for all Vκ 1-6, VH1-7, Vλ1-11, nine CH isotypes, Cκ, Cκ, Cλ1 &3, FcεRI α,β, and γ subunits, all three FcγR and their subunits, and FcαR. Validating this on a volunteer PBMC cDNA, we show a qPCR primer set repertoire that can quantify the relative expression of all the above genes to GAPDH housekeeping gene, with implications and uses in both clinical monitoring and research.