Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to "absolute quantification", which offers the potential for reliable value assignments of DNA reference materials, and International System of Units (SI) traceability to copy number 1 through accurate counting. In this study, 2 enumeration-based methods, flow cytometric (FCM) counting and the digital polymerase chain reaction (dPCR), were compared to quantify a solution of the pBR322 plasmid at a concentration of several thousand copies per microliter. In addition, 2 orthogonal chemical-analysis methods based on nucleotide quantification, isotope-dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) were applied to quantify a more concentrated solution of the plasmid. Although 9 dPCR results from 8 laboratories showed some dispersion (relative standard deviation [RSD] = 11.8%), their means were closely aligned with those of the FCM-based counting method and the orthogonal chemical-analysis methods, corrected for gravimetric dilution factors. Using the means of dPCR results, the RSD of all 4 methods was 1.8%, which strongly supported the validity of the recent enumeration approaches. Despite a good overall agreement, the individual dPCR results were not sufficiently covered by the reported measurement uncertainties. These findings suggest that some laboratories may not have considered all factors contributing to the measurement uncertainty of dPCR, and further investigation of this possibility is warranted.
This is the first phylogenetic analysis integrating both morphological and molecular data of the sponge suborder Mycalina (Poecilosclerida), which was erected in 1994. A cladistic analysis of morphology supported the monophyly of Cladorhizidae (including Euchelipluma), Guitarridae (excluding Euchelipluma), Isodictyidae, Latrunculiidae, and Podospongiidae but rejected monophyly for Desmacellidae, Esperiopsidae, Hamacanthidae, and Mycalidae. Analyses of partial 16S and partial 28S rRNA datasets combined, as well as that of a complete 18S rDNA dataset, suggest that Mycalina is not monophyletic; Biemnidae is only distantly related to other poecilosclerids; Merlia and Desmacella branch near the base of a diverse Poecilosclerida clade; Mycalidae is monophyletic (excluding Mycale [Anomomycale] titubans in 18S); and Esperiopsidae and Isodictyidae form a clade. Analyses of the two molecular datasets differed on the monophyly of Podospongiidae and about the relationship of Podospongiidae to Isodictyidae + Esperiopsidae.
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