Genomic DNA Extraction from Different Human Tissue Sample: Urine, Oral Gargle, Blood, and Cervical for Real Time Amplification by qPCR

Molecular technologies have been a driver of rapid paradigm shift in the field of scientific research and clinical applications. Adequate and pure form of nucleic acid extracted from efficient biological sources together with an easy, simple, standardized protocol, ease of access and acceptability is also a prerequisite for genotyping analysis for different disorders. Urine and oral gargle samples are emerging as a potential source of genomic DNA (gDNA) and may offer a better alternative to existing sources such as blood. This manuscript compares for the first time, the quality and quantity of gDNA extracted manually by standard methods from different biological samples like urine, oral gargle, blood and cervical samples, together. It aimed to assess the feasibility of extracting sufficient gDNA from easily accessible and acceptable samples for amplification. 646 urine, oral, blood and cervical samples were collected from different studies in the department and analyzed. gDNA from blood was extracted by Proteinase K digestion followed by ethanol precipitation, and phenol-chloroform extraction method for urine, oral gargle and cervical specimen. The quantification of isolated gDNA were analyzed in Nano-drop spectrophotometer and 1% Agarose gel electrophoresis for different sample types. Human beta-globin gene were used for internal quality control for the real time amplification. DNA quantity and purity were adequate and comparable for amplification in all the three biological samples with an average A 260/280 ratio of 1.8, 1.7 and 1.7 for gDNA isolated from urine, oral gargle and blood samples, respectively (n = 200 each). The mean DNA yield from urine, oral and blood samples were 474.9, 899.2 and 489.0 (ng/mu l) respectively. The mean concentration of the DNA extracted from cervical smear samples (n = 46) was found to be 318.6 ng/mu l with an average A 260/280 of 1.6. The average Cq values obtained were 12.8, 18.5 and 17.9 for beta-globin in gDNA isolated from urine, oral and blood samples respectively (n = 50 each). The present study concluded that adequate and pure form of gDNA can be extracted from different biological samples of blood, urine and oral samples using standardized manual DNA extraction protocol. The extracted gDNA was amplified for gene targets as per respective study objectives. Detection of beta-globin, IC with Cq cut off value below 30 for adequate internal quality. Genomic DNA extraction was successful from cervical tissue, however, could not be used for like-to-like comparison as it requires further work to elicit successful amplification.