title: Quantification of type II procollagen splice forms using alternative transcript-qPCR (AT-qPCR).
abstract: During skeletal development, the onset of chondrogenic differentiation is marked by expression of the α1(II) procollagen (Col2a1) gene. Exon 2 of Col2a1 codes for a cysteine-rich von Willebrand factor C-like domain. Chondroprogenitors express the exon 2-containing IIA and IID splice forms by utilizing adjacent 5' splice sites separated by 3 base pairs. There is a shift to expression of the shorter, exon 2-lacking IIB splice form with further differentiation. Alternative splicing analysis of Col2a1 splice forms has often relied upon semi-quantitative PCR, using a single set of PCR primers to amplify multiple splice forms. We show that this widely used method is inaccurate due to mismatched amplification efficiency of different-sized PCR products. We have developed the TaqMan®-based AT-qPCR (Alternative Transcript-qPCR) assay to more accurately quantify alternatively spliced mRNA, and demonstrate the measurement of Col2a1 splice form expression in differentiating ATDC5 cells in vitro, and in wild type mouse embryonic and postnatal cartilage in vivo. The AT-qPCR assay is based on the use of a multiple-amplicon standard (MAS) plasmid, containing a chemically synthesized cluster of splice site-spanning PCR amplicons, to quantify alternative splice forms by standard curve-based qPCR. The MAS plasmid designed for Col2a1 also contained an 18S rRNA amplicon for sample normalization, and an amplicon corresponding to a region spanning exon 52-53 to measure total Col2a1 mRNA. In mouse E12.5 to P70 cartilages, we observed the expected switch between the IIA and IIB splice forms; no IID or IIC splice products were observed. However, in the ATDC5 cultures, predominant expression of the IIA and IID splice forms was found at all times in culture. Additionally, we observed that the sum of the IIA, IIB and IID splice forms comprises only a small fraction of Col2a1 transcripts containing the constitutive exon 52-53 junction. We conclude from our results that the majority of ATDC5 cells in the assay described in this study remained as chondroprogenitors during culture in standard differentiation conditions, and that additional Col2a1 transcripts may be present. The validity of this novel AT-qPCR assay was confirmed by demonstrating the expected Col2a1 isoform expression patterns in vivo in developing mouse cartilage. The ability to measure true levels of procollagen type II splice forms will provide better monitoring of chondrocyte differentiation in other model systems. In addition, the AT-qPCR assay described here could be applied to any gene of interest to detect and quantify known and predicted alternative splice forms and can be scaled up for high throughput assays.
authors.affiliation: Department of Orthopaedic Surgery, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, United States.
authors.forename: Audrey
authors.initials: A
authors.lastname: McAlinden
authors.forename: Kyu-Hwan
authors.initials: KH
authors.lastname: Shim
authors.forename: Louisa
authors.initials: L
authors.lastname: Wirthlin
authors.forename: Soumya
authors.initials: S
authors.lastname: Ravindran
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Hering
mesh_terms: D000818:Animals; D001483:Base Sequence; D020219:Chondrogenesis / Q000235:genetics / Q000502:physiology*; D024043:Collagen Type II / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D005455:Fluorescent Antibody Technique; D018507:Gene Expression Regulation, Developmental / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D020033:Protein Isoforms / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D019914:Taq Polymerase
chemical_list: C499177:COL2A1 protein, human; D024043:Collagen Type II; D018076:DNA, Complementary; D020033:Protein Isoforms; D019914:Taq Polymerase