METHODS: In this study, mouthwash, saliva, and buccal cytobrush samples were collected from β-thalassemia major patients who had previously been characterized using DNA extracted from peripheral blood. DNA was extracted from mouthwash, saliva, and buccal cytobrush samples using the conventional inexpensive phenol-chloroform method and was measured by spectrophotometry for yield and purity. Molecular characterization of β-globin gene mutations was carried out using the amplification refractory mutation system (ARMS).
RESULTS: DNA extracted from mouthwash, saliva, and buccal cytobrush samples produced high concentration and pure DNA. The purified DNA was successfully amplified using ARMS. Results of the β-globin gene mutations using DNA from the three non-invasive samples were in 100% concordance with results from DNA extracted from peripheral blood.
CONCLUSIONS: The conventional in-house developed methods for non-invasive sample collection and DNA extraction from these samples are effective and negate the use of more expensive commercial kits. In conclusion, DNA extracted from mouthwash, saliva, and buccal cytobrush samples provided sufficiently high amounts of pure DNA suitable for molecular analysis of β-thalassemia.
METHODS: A total of 322 samples of mainly human origin were analysed using eight protocols, applying a wide variety of laboratory components. Several samples (60% of human specimens) were processed using different protocols. In total, 712 sequencing libraries were investigated for viral sequence contamination.
RESULTS: Among sequences showing similarity to viruses, 493 were significantly associated with the use of laboratory components. Each of these viral sequences had sporadic appearance, only being identified in a subset of the samples treated with the linked laboratory component, and some were not identified in the non-template control samples. Remarkably, more than 65% of all viral sequences identified were within viral clusters linked to the use of laboratory components.
CONCLUSIONS: We show that high prevalence of contaminating viral sequences can be expected in HTS-based virome data and provide an extensive list of novel contaminating viral sequences that can be used for evaluation of viral findings in future virome and metagenome studies. Moreover, we show that detection can be problematic due to stochastic appearance and limited non-template controls. Although the exact origin of these viral sequences requires further research, our results support laboratory-component-linked viral sequence contamination of both biological and synthetic origin.
MATRIALS AND METHODS: The conventional protocol and three microwave protocols of tissue processing were used in this study. A pilot study was done prior to the real run to determine the baseline timing for microwave protocol. The baseline timing was fixed at 2 minutes,30 minutes,5 minutes and 25 minutes. The processing time of the microwave protocol was adjusted from 62 minutes to 70 minutes to 77 minutes by increasing the dehydration and wax impregnation time while the time for tissue fixation and clearing remain the same throughout all the microwave protocols.
RESULTS: The group 2 microwave protocol produced the sections that is closely comparable to group 1 conventional protocol. The morphological quality of histopathology slides is best observed when the processing time of microwave protocol is 62 minutes.
CONCLUSION: The most appropriate microwave protocol for tissue processing is group 2 as the morphological quality of histopathology slides are more superior than that of group 1 with an overall percentage of 80% of satisfactory slides in group 2 and 76.68% in group 1.