Standardization of<i>TP53</i>gene mutations analysis on oral squamous cell carcinoma from paraffin-embedded tissues

Júnior, José de Assis Silva; Camisasca, Danielle Resende; Tavares, Débora dos Santos; Faria, Paulo Antônio Silvestre de; Dias, Fernando Luiz; Ribeiro, Georgina Severo; Amorim, Lı́dia Maria da Fonte de; Lourenço, Simone De Queiroz Chaves

doi:10.5935/1676-2444.20140009

Cited by 1 publication

(4 citation statements)

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“…Hematoxylin-eosin staining was performed, and the slides were observed under a light microscope (Olympus, Tokyo, Japan) by a pathologist to mark the cancer area, which would serve as the guide for the area of interest in the other non-stained slides. 15,24,25 The marked area was transferred onto paper. The tissue was cut to a thickness of 3 μm into five non-stained slides and placed on a hot plate briefly for initial deparaffinization 19 and to allow the tissue to adhere well to the slides.…”

Section: Methodsmentioning

confidence: 99%

“…The slides were then aligned to overlap the previously marked area on the paper, and the microdissection of the cancer area was performed using disposable scalpels. 24 The microdissected tissues (0.1 ml) were stored in 1.5 ml microtubes at room temperature prior to the DNA extraction process. Some samples with a large area of cancer required two non-stained slides for microdissection to reach the 0.1 ml volume.…”

Section: Methodsmentioning

confidence: 99%

“…DNA extraction was conducted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany), and all the methods followed the manufacturer's manual, 19,24 except for the proteinase K incubation step. Initially, the samples were washed with xylene to dissolve the excess paraffin, followed by two washes with absolute ethanol.…”

Section: Methodsmentioning

confidence: 99%

“…These findings led the authors to prolong the incubation period to 48 hours at room temperature, which is the minimal activation temperature for proteinase K to be active, 23 and four hours at 56ºC, the optimum temperature for proteinase K, 22 to ensure complete tissue lysis. Thus, the aim of this study is to find the most optimized duration of proteinase K incubation protocols to achieve the highest DNA yields: one-hour incubation at 56ºC as per the manufacturer's instructions, 24-hour incubation at 56ºC, 19 and 48 hours at room temperature with an additional four hours at 56ºC.…”

Section: Introductionmentioning

confidence: 99%

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Optimization of proteinase K incubation protocol duration during DNA extraction from oral squamous cell carcinoma FFPE samples

Meizarini,

Puteri,

Yasan

et al. 2023

Dent. J.

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Background: Formalin-fixed paraffin-embedded (FFPE) specimen archives are a valuable source of sample material for molecular biological analysis. However, the DNA isolated from FFPE samples is usually low in concentration and fragmented. Thus, it is necessary to optimize the FFPE DNA extraction protocol to obtain the best results. Proteinase K incubation is undoubtedly crucial in DNA extraction procedures, but this step is often not well explained in the manufacturer’s manual. Purpose: This study aimed to find the optimal duration for proteinase K incubation protocols to achieve the highest DNA yields. Methods: Fifteen paraffin blocks of Oral Squamous Cell Carcinoma (OSCC) specimens were obtained, and the cancerous areas were microdissected into smaller cuts for DNA extraction. The samples were randomly divided into three groups (n=5) and subjected to three different proteinase K incubation protocols: one-hour incubation at 56ºC as per the manufacturer’s instructions (Group I), 24-hour incubation at 56ºC (Group II), and 48 hours at room temperature with an additional four hours at 56ºC (Group III). The extracted DNA was then quantified using a Nanodrop spectrophotometer. The recorded data were analyzed using ANOVA-LSD. Results: The highest DNA concentration was found in Group III (107.74 ± 41.92), which was significantly higher compared to Group II (59.46 ± 30.32) and Group I (6.46 ± 1.97) (p<0.05). Conclusion: In conclusion, modifying the duration of proteinase K incubation protocols can lead to different DNA yield results. In this study, the most optimized protocol for proteinase K incubation, resulting in the highest DNA yields, was 48 hours at room temperature with an additional four hours at 56ºC.

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