Department of Orthopaedic Surgery, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, United States.
Matrix Biol. 2012 Sep-Oct;31(7-8):412-20. doi: 10.1016/j.matbio.2012.08.002. Epub 2012 Sep 10.
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.
在骨骼发育过程中，软骨分化的开始标志着α1(II)前胶原（Col2a1）基因的表达。Col2a1 的外显子 2 编码富含半胱氨酸的血管性血友病因子 C 样结构域。软骨祖细胞通过利用相邻的 3 个碱基对分隔的 5' 剪接位点来表达包含外显子 2 的 IIA 和 IID 剪接形式。随着进一步分化，会出现表达较短、缺少外显子 2 的 IIB 剪接形式的转变。Col2a1 剪接形式的选择性剪接分析通常依赖于半定量 PCR，使用一组 PCR 引物来扩增多种剪接形式。我们表明，由于不同大小的 PCR 产物的扩增效率不匹配，这种广泛使用的方法是不准确的。我们开发了基于 TaqMan®的 AT-qPCR（替代转录物-qPCR）测定法，以更准确地定量替代剪接的 mRNA，并证明了在体外分化的 ATDC5 细胞中以及在体内野生型小鼠胚胎和出生后软骨中测量 Col2a1 剪接形式表达的方法。AT-qPCR 测定法基于使用多扩增子标准（MAS）质粒，该质粒包含化学合成的剪接位点跨越 PCR 扩增子簇，通过基于标准曲线的 qPCR 定量替代剪接形式。为 Col2a1 设计的 MAS 质粒还包含用于样品归一化的 18S rRNA 扩增子，以及跨越外显子 52-53 测量总 Col2a1 mRNA 的扩增子。在 E12.5 到 P70 天的小鼠软骨中，我们观察到 IIA 和 IIB 剪接形式之间的预期转变；没有观察到 IID 或 IIC 剪接产物。然而，在 ATDC5 培养物中，在整个培养过程中始终发现 IIA 和 IID 剪接形式的主要表达。此外，我们观察到 IIA、IIB 和 IID 剪接形式的总和仅占含有组成型外显子 52-53 连接的 Col2a1 转录本的一小部分。我们从研究结果中得出结论，在本研究中描述的测定法中，大多数 ATDC5 细胞在标准分化条件下培养期间仍然作为软骨祖细胞存在，并且可能存在其他 Col2a1 转录本。通过在体内发育中的小鼠软骨中证明预期的 Col2a1 同工型表达模式，验证了这种新型 AT-qPCR 测定法的有效性。能够测量真正的 II 型前胶原剪接形式水平将为其他模型系统中的软骨细胞分化提供更好的监测。此外，这里描述的 AT-qPCR 测定法可以应用于任何感兴趣的基因，以检测和定量已知和预测的替代剪接形式，并可以扩展用于高通量测定法。
