Our proof-of-concept multiplex hextuple luciferase assay was designed to simultaneously monitor the p53, TGF-β, NF-κβ, c-Myc, and MAPK/JNK signaling pathways.
Because all six reporters are on a single piece of DNA, a single vector ensures stoichiometric ratios of each transcriptional unit in each transfected cell, resulting in lower experimental variation. We used synthetic assembly cloning ( Vazquez-Vilar, et al., 2020) to assemble all six luciferase reporter units into a single vector, over eight stitching rounds. We recently developed a multiplex luciferase assay that allows monitoring the activity of five experimental pathways against one control simultaneously.
They are genetically encoded, versatile, and cost-effective, whose output signals can be sensitively detected. Luciferases are good candidates for generation of such signals. The signals from such measurements should be independently detectable and measure large dynamic ranges. While most cell-based assays measure single quantities, multiplexed assays seek to address these limitations by obtaining multiple simultaneous measurements. Such assays are designed to examine the effects of small compounds on targets, pathways, or phenotypes participating in normal and disease processes. Hence, awareness for uptake of cancer screening and early detection are encouraged.High-throughput cell-based screening assays are valuable tools in the discovery of chemical probes and therapeutic agents. Our study also revealed the poor knowledge of Nigerian women to cancer screening, this may be one of the contributory factors to late diagnosis of cervical cancer among the African population. The histopathological result, alongside the Multiple Sequence Alignment revealed that with higher severity of cervical carcinoma, the p53 gene tends to accumulate more mutations. Sociodemographic characteristics revealed that 60% of the study participants has husbands with multiple sexual partners and that only 23.3% of the participants have ever done pap smear test prior to diagnosis, while 20% of them are unaware of the screening test. The outcome of this study showed nineteen identified polymorphic variants Missense mutations occurred in 47% of the samples, 32% were silent mutations, 16% were frameshift mutation and 5% nonsense mutation. Multiple Sequence Alignment using T-COFFEE was performed to reveal the polymorphic variations in the samples. Nucleotide BLAST was performed on the DNA sequence for sequence identity and retrieval of reference sequence. The PCR products were sequenced at Inqaba Biotec, and chromatogram was analyzed using FinchTv and SnapGene Viewer. The region of interest was amplified using Polymerase Chain Reaction (PCR), this was quantified on a 1.5% agarose gel, and the ethidium bromide stained gel was viewed on a transilluminator. The purified genomic DNA was quantified using NanoDropTM 2000 spectrophotometer. Genomic DNA was isolated and purified from cervical tissue biopsies obtained from patients reporting at the University College Hospital, Ibadan. This research study explored the mutations in the exon 3 and exon 4 of the P53 gene in cervical cancer. All subjects Allied Health Cardiology & Cardiovascular Medicine Dentistry Emergency Medicine & Critical Care Endocrinology & Metabolism Environmental Science General Medicine Geriatrics Infectious Diseases Medico-legal Neurology Nursing Nutrition Obstetrics & Gynecology Oncology Orthopaedics & Sports Medicine Otolaryngology Palliative Medicine & Chronic Care Pediatrics Pharmacology & Toxicology Psychiatry & Psychology Public Health Pulmonary & Respiratory Medicine Radiology Research Methods & Evaluation Rheumatology Surgery Tropical Medicine Veterinary Medicine Cell Biology Clinical Biochemistry Environmental Science Life Sciences Neuroscience Pharmacology & Toxicology Biomedical Engineering Engineering & Computing Environmental Engineering Materials Science Anthropology & Archaeology Communication & Media Studies Criminology & Criminal Justice Cultural Studies Economics & Development Education Environmental Studies Ethnic Studies Family Studies Gender Studies Geography Gerontology & Aging Group Studies History Information Science Interpersonal Violence Language & Linguistics Law Management & Organization Studies Marketing & Hospitality Music Peace Studies & Conflict Resolution Philosophy Politics & International Relations Psychoanalysis Psychology & Counseling Public Administration Regional Studies Religion Research Methods & Evaluation Science & Society Studies Social Work & Social Policy Sociology Special Education Urban Studies & Planning BROWSE JOURNALS
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