Comparative evaluation of DNA synthesis for qPCR analysis from oral squamous cell carcinoma tissues - A rapid and robust isolation technique for gene expression studies

DNA isolation from biological materials is the initial step in several bioanalytical processes, including the polymerase chain reaction (PCR). This procedure works best with pure DNA devoid of potential amplification reaction inhibitors. Since the quantity and quality of biological samples are limited, it is essential to extract as much DNA as possible from the original sample. The solid-phase extraction technique is frequently used to purify DNA. In this process, the DNA is adsorbed onto a solid support, any contaminants from the reaction are eliminated by washing, and the purified DNA is then eluted from the support. The quality and concentration of DNA have a direct effect on the gene analysis procedure. Therefore, before interpreting the results of PCR-based diagnostic assays, it is imperative to confirm the DNA preparation and integrity, particularly if no pathogenic DNA is identified. A comparative study was carried out using two standardized protocols for DNA synthesis in comparison with a test protocol incorporating carrier RNA and proteinase K from a viral detection kit. The quality and quantity of the synthesized DNA are assessed by qPCR analysis for gene amplification of metastasis suppressor genes NDRG1, RhoGDI, and KISS 1. The test protocol showed higher yields of DNA in comparison to the standard protocol. The concentration of DNA obtained was validated by the concentration of gene expressed from q PCR analysis. The gene expression was significantly higher when the test protocol method was followed for DNA synthesis. Thus the study validates the potential of nucleic acid carrier RNA molecules to improve DNA extraction processes used in tissue samples for gene analysis procedures. • The technique of quantitative polymerase chain reaction (qPCR) has revolutionized molecular biology research and has become an essential tool in various fields, including diagnostics, genomics, and gene expression analysis. • qPCR relies on the amplification of a specific DNA target using PCR, followed by quantification of the amplified product. The accuracy and reliability of the qPCR results heavily depend on the efficiency of DNA synthesis during the amplification step. • The current study focuses on finding the most reliable technique for DNA synthesis in comparison to the standard protocol. • The introduction of Proteinase K and Carrier RNA plays a vital role extracting high quality DNA for gene expression studies. • The current study validated the production of high-quality DNA by amplifying the target genes using carrier RNA which can be used for further studies. It is proven to be the superior method for gene amplification.