Corrigendum to “Quantification of type II procollagen splice forms using alternative transcript-qPCR (AT-qPCR)” (Matrix Biol. 31(2012) (412-420)(S0945053X12000911)(10.1016/j.matbio.2012.08.002))

Audrey McAlinden, Kyu Hwan Shim, Louisa Wirthlin, Soumya Ravindran, Thomas M. Hering

Research output: Contribution to journalComment/debate

Abstract

Our original published paper describes a novel method developed in our laboratories for the determination of the relative proportions of individual mRNA alternative splice-forms. The paper contained a detailed protocol describing the assay method, and subsequent calculations. It is universally applicable to any mRNA gene product having multiple spice forms. To show the utility of the method, we presented an analysis of changes in the proportions of Col2a1 splice variants during in-vitro chondrogenesis of ATDC5 cells grown in culture, as well as an analysis of embryonic and postnatal mouse epiphyseal cartilage. To our knowledge, there are no errors in the assay method as presented in the original paper. We discovered that the results of our demonstration assays, however, were flawed, and therefore we submit this corrigendum to rectify this problem. Specifically, the authors regret to report here that an error was made in serial dilution of the multiple-amplicon standard (MAS) plasmid used to plot standard curves to quantify Col2a1 alternative splice-forms by “absolute” qPCR. Our intention was to produce a ten-fold dilution series. Inadvertently, a twenty-five-fold dilution series was made, but all subsequent calculations assumed ten-fold dilutions. The incorrect slopes of MAS standard curves resulted in miscalculation of splice form proportions. In this corrigendum, beginning with the originally presented raw data, we have performed the required recalculations and have modified tables and figures throughout the paper according to the recalculated results. We have replaced Fig. 5 which shows our analysis of Col2a1 splice forms during ATDC5 differentiation, as well as Fig. 6, which shows our analysis of splice forms in embryonic and postnatal mouse epiphyseal cartilage. We have changed Fig. 7, which demonstrates the major errors inherent to the often-used co-amplification method for quantifying splice forms. Fortunately, the major conclusions of this study are unchanged. We wish to mention two changed results and their significance. First, we have now determined that the maximum expression of the IIB splice form during ATDC5 chondrogenesis is actually ~29%, rather than ~4% as reported in our paper. Therefore, our conclusion that IIB is not the predominant transcript after 4 weeks of culture in this system remains valid. Secondly, after recalculating our data, the ratio of total Col2a1 mRNA to the sum of individually measured splice forms at all time points was determined to be consistently ~2, rather than 76 to 145-fold reported originally (Fig.5). Therefore, alternative transcripts other than the IIA, IIB, IIC and IID forms may still be present, but at much lower levels. As we previously concluded, we now find that at all time points the order of splice form abundance in ATDC5 cultures was IID>IIA>IIB>IIC. In recalculating our data, we confirmed our evidence that transcripts other than IIA, IIB, IIC, and IID are present, but in larger amounts that shown in Fig. 6, with the ratio varying considerably in embryonic and postnatal cartilage over time. We have provided corrected text, figures, tables from the paper and previous supplementary material in the online supplement to this corrigendum. Spreadsheets showing detailed calculations are provided as Microsoft Excel files, which can be used as templates for calculations by other investigators using our method. The authors would like to apologize for any inconvenience caused by the error in our paper, and for the delay in preparation of this corrigendum. The authors also wish to indicate that the errors described in this corrigendum were discovered and corrected prior to the publication of another paper from our laboratory using the AT-qPCR method and the same murine MAS plasmid described here (Hering, T.M., et al, Matrix Biology 36 (2014) 51-63.

Original languageEnglish
Pages (from-to)119-120
Number of pages2
JournalMatrix Biology
Volume65
DOIs
StatePublished - Jan 2018

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