title: Absolute quantification of gene expression in biomaterials research using real-time PCR.
abstract: 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.
authors.affiliation: Department of Biological Sciences, National University of Singapore, Republic of Singapore.
authors.forename: David Tai
authors.initials: DT
authors.lastname: Leong
authors.forename: Anurag
authors.initials: A
authors.lastname: Gupta
authors.forename: Hui Fen
authors.initials: HF
authors.lastname: Bai
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Wan
authors.forename: Li Foong
authors.initials: LF
authors.lastname: Yoong
authors.forename: Heng-Phon
authors.initials: HP
authors.lastname: Too
authors.forename: Fook Tim
authors.initials: FT
authors.lastname: Chew
authors.forename: Dietmar Werner
authors.initials: DW
authors.lastname: Hutmacher
mesh_terms: D001672:Biocompatible Materials / Q000378:metabolism*; D045744:Cell Line, Tumor; D017931:DNA Primers; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism
chemical_list: D001672:Biocompatible Materials; D017931:DNA Primers; D012333:RNA, Messenger