Leong David Tai, Gupta Anurag, Bai Hui Fen, Wan Guoqiang, Yoong Li Foong, Too Heng-Phon, Chew Fook Tim, Hutmacher Dietmar Werner
Department of Biological Sciences, National University of Singapore, Republic of Singapore.
Biomaterials. 2007 Jan;28(2):203-10. doi: 10.1016/j.biomaterials.2006.09.011. Epub 2006 Oct 10.
One major measurement of tissue-engineered constructs efficacy and performance is determining expression levels of genes of interest at the molecular level. This measurement is commonly carried out with reverse transcription-polymerase chain reaction (RT-PCR). In this study, we described a novel method in achieving absolute quantification of gene expression using real-time PCR (aqPCR). This novel method did not require molecular cloning steps to prepare the standards for quantification comparison. Standards were linear double-stranded DNA molecules instead of the typical gene-in-plasmid format. aqPCR could also be used to give relative quantification comparisons between samples simply by dividing the copy numbers readings of the gene of interest with that of the normalization gene. RNA was extracted from monolayer and from polycaprolactone scaffold cultures and assayed for beta-actin and osteocalcin genes. We compared our aqPCR method with end-point PCR since end-point PCR is still a common means of measuring gene expression in the biomaterials field. This study showed that aqPCR was a better method to quantify gene expression than end-point PCR. With our described linear DNA standards method, we were able to obtain not only relative quantification of osteocalcin and beta-actin expression level but also actual copy numbers of osteocalcin and beta-actin for the monolayer culture and to be 1.34 x 10(4) and 1.45 x 10(7) copies, respectively and for the scaffold cultures to be 772 and 2.83 x 10(5) copies, respectively per starting total RNA mass of 10 ng. The standards curves made from these linear DNA standards showed good linearity (R(2)=0.9964 and 0.9902 for osteocalcin and beta-actin standards graphs), ranged from 10 to 10(9) copies and of comparable accuracy to current absolute quantification real-time PCR methods (which used plasmid standards obtained through molecular cloning methods). Our method might be a viable and more user-friendly alternative to current absolute quantification real-time PCR protocols.
组织工程构建体功效和性能的一项主要测量指标是在分子水平上确定感兴趣基因的表达水平。这种测量通常通过逆转录-聚合酶链反应（RT-PCR）来进行。在本研究中，我们描述了一种使用实时定量PCR（aqPCR）实现基因表达绝对定量的新方法。这种新方法不需要分子克隆步骤来制备用于定量比较的标准品。标准品是线性双链DNA分子，而不是典型的质粒载体中的基因形式。aqPCR还可用于通过将感兴趣基因的拷贝数读数除以标准化基因的拷贝数读数，简单地给出样品之间的相对定量比较。从单层培养物和聚己内酯支架培养物中提取RNA，并检测β-肌动蛋白和骨钙素基因。我们将我们的aqPCR方法与终点PCR进行了比较，因为终点PCR仍然是生物材料领域中测量基因表达的常用方法。这项研究表明，aqPCR是一种比终点PCR更好的基因表达定量方法。通过我们描述的线性DNA标准品方法，我们不仅能够获得骨钙素和β-肌动蛋白表达水平的相对定量，还能够获得单层培养物中骨钙素和β-肌动蛋白的实际拷贝数，分别为1.34×10⁴和1.45×10⁷拷贝，对于支架培养物，每10 ng起始总RNA质量分别为772和2.83×10⁵拷贝。由这些线性DNA标准品制成的标准曲线显示出良好的线性（骨钙素和β-肌动蛋白标准曲线的R²分别为0.9964和0.9902），范围从10到10⁹拷贝，并且与当前的绝对定量实时PCR方法（使用通过分子克隆方法获得的质粒标准品）具有相当的准确性。我们的方法可能是当前绝对定量实时PCR方案的一种可行且更用户友好的替代方法。
