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 β-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 β-actin expression level but also actual copy numbers of osteocalcin and β-actin for the monolayer culture and to be 1.34×10⁴ and 1.45×10⁷ copies, respectively and for the scaffold cultures to be 772 and 2.83×10⁵ 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 β-actin standards graphs), ranged from 10 to 10⁹ 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.

Introduction

When analyzing tissue-engineered constructs or studying biomaterials–cells interactions, it is almost a certainty to quantify performance of such constructs in terms of in vitro and in vivo characteristics. Measurements are usually made in the macroscopic and microscopic level. However, in order to complete the characterization, it is often necessary to measure performance of these constructs at the molecular level. It comes as no surprise that recent papers in the biomaterials and biomedical fields used gene expressions of cells and their responses to their 3D environments [1], [2], [3], [4], [5] to measure the performance of these tissue-engineering constructs.

Reverse transcription-polymerase chain reaction (RT-PCR) is an in vitro method for enzymatically amplifying (indirectly) defined sequences of messenger RNA (mRNA) [6]. This method is sensitive enough to compare mRNA from as little as one cell [7]. RT-PCR can be used to compare levels of mRNA in different sample populations, to characterize patterns of mRNA expression and to discriminate between closely related mRNAs [8]. With this immense ability to amplify rare copies of cDNA, PCR has opened up another level of sophistication in ascertaining gene expression in cells–environment interactions.

RT-PCR can be carried out in two ways, end-point PCR and real-time PCR. The major difference between these two methods is that for end-point PCR, quantification takes place at the end of the entire PCR reactions while real-time PCR takes measurements at the exponential phase of the PCR.

There are two common categories of quantification using real-time RT-PCR, (a) relative and (b) absolute quantification.

Relative quantification is commonly carried by normalization of the expression levels of the gene of interest with that of the housekeeping genes. Relative quantification with real-time PCR has been used in comparing osteogenic genes in bone marrow-cancellous bone [9]; chondrogenic gene expression of bovine chondrocytes seeded on hybrid PLGA mesh with collagen-filled pores [10]. Relative quantification however would only give a ratio of the gene of interest comparison and not actual copy numbers in a defined concentration of mRNA population. Useful biological information might be lost with using the ratio. Since a high ratio may not necessarily mean a high expression of the gene of interest as the ratio is sensitive to the expression level of the normalizing gene (the denominator).

Absolute quantification relies on a standards plot constructed from the known concentrations of standards template and corresponding levels of real-time PCR data. Commonly, standards are derived from purified plasmid dsDNA, in vitro-transcribed RNA or in vitro-synthesized ssDNA [11]. The amount is quantified spectroscopically at 260 nm or with DNA fluorescent dye [12] and converted to number of copies using the molecular weight of the RNA or DNA sequence. Actual copy numbers of the gene of interest in the samples could then be read off the standards plot. Absolute quantification therefore would eliminate the ambiguous use of ratios.

However, the current methods of producing standards for the absolute quantification real-time PCR can be technically tedious since it often involves molecular cloning of the target sequence into vector systems and amplification in Escherichia coli. In this study, we introduced another method to quantify actual copies of starting mRNA using real-time PCR technique but did not require molecular cloning steps to obtain the standards. This method used linear double-stranded (ds) DNA standards purified from PCR reactions containing the target sequence. mRNA from positive control cells was first reversed transcribed to cDNA and then with appropriately designed primers, the target sequence was amplified with standard PCR methods. The correct PCR products were confirmed using standard gel electrophoresis and isolated from the gel band. The PCR products could be further sequenced for further confirmation of the sequence. The purified ds DNA were then quantified and serially diluted to produce a standards graph. For each assaying routine, the samples were also subjected to the same conditions as the standards. The standards readings were used to plot a standard plot and the actual copy numbers of the samples could then be read off the plot.

This technique could be easily adapted to existing real-time PCR protocols. We also discussed on the advantages and limitations of the described aqPCR method.