Development of a Universal Taqman Probe for mRNA Gene Expression Analysis

The detection and monitoring of eukaryotic gene expression have become integral aspects of biological control assessment and diagnosis or treatment of diseases. Taqman probe and SYBR Green I are two most popular fluorescent reporter molecules in gene expression assays, with Taqman probes becoming the preferred choice for real-time PCR as they only detect the targeted product, although a new probe must be synthesized for each target. Here, we present a novel and specific mRNAs quantification method using an attached stem-loop reverse transcriptase universal template and probe. In this method, different target mRNA sequences can be detected employing the same universal probe. In addition, it requires a small region (at least 35 nucleotides) of the gene for primer design, therefore, facilitating the design of primer especially for highly polymorphic regions. The feasibility and robustness of this approach were studied and compared to SYBR Green I for adenosine deaminase (ADA), ornithine decarboxylase (ODC) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes in MCF-7 cell line treated with Achillea millefolium extract. Results show that ADA and ODC were overexpressed following treatment with different concentrations of Achillea millefolium in universal Taqman assays but not in SYBR Green I assays. Universal Taqman probe assay is an easy, specific and efficient qPCR method allowing effective profiling of many mRNAs without microarray and expensive platforms.