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Document Type |
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Thesis |
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Document Title |
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REUSE OF DNA EXTRACTED FROM ALREADY IMMUNOSTAINED FORMALIN FIXED PARAFFIN EMBEDDED (FFPE) SLIDES: TECHNIQUE REFINEMENT AND USE IN DOWNSTREAM MOLECULAR ANALYSIS TO STUDY CANCER BIOLOGY إعادة استخدام الحمض النووي المستخرج من عينات الصبغة المناعية المستعملة من الشرائح الشمعية: تحسين تقنية الاستخراج والاستخدام في التحاليل الجزيئية لدراسة بيولوجيا السرطان |
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Subject |
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Faculty of Sciences |
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Document Language |
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Arabic |
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Abstract |
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So far, the conventional (FFPE) Formalin Fixed Paraffin Embedded technique is the gold standard for preserving histomorphology, while cryopreservation of tissue is the gold standard for biomolecules’ preservation. Once these tissues stained, the slides are routinely archived along with FFPE blocks for long in biobanks/hospitals. However, the reuse of already fixed and stained biospecimens to extract DNA is not a common practice worldwide. This study focused on the feasibility of extracting DNA from already immunohistochemistry (IHC) stained slides to assess its quality and usefulness for further downstream molecular analysis and validation at genetic level. DNA was extracted and reused to evaluate the mutational profile of selected proto-oncogene KRAS, which its protein plays a crucial role in normal tissue signaling, and its mutation is a vital footstep in the development and progression of many cancer types such as colorectal carcinoma (CRC). With the shortage in high-quality and fully annotated biospecimens, we believe that this technique will improve our best practices associated with the use of biospecimens in biorepositories and medical research. FFPE IHC slides from CRC consented patients were made and stored in the CEGMR Biobank, King Abdulaziz University. A workflow designed by our group consists successively of removing the slide cover-slip by applying 44 pipelines, which were a combination of 11 chemicals, temperature (room temperature versus 37°C) and shaking (shaking versus no shaking). Seven pipelines that showed a cover-slip removal time around 24 to 30 hours were selected for the study, then the non-covered FFPE slides were followed by crude dissection of the IHC stained tissue and DNA extraction using the QIAamp DNA FFPE Tissue Kit according to manufacturer's instructions . After DNA quality assessment, downstream mutational analysis of KRAS was analysed. A high quality DNA was obtained allowing KRAS gene sequencing patterns, clean DNA chromatograms with evenly-spaced peaks, each with only one color were achieved. The study suggested a promising technique to reuse DNA from IHC stained slides with obtaining a suitable DNA concentration and integrity as starting biomolecule for further downstream molecular applications. |
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Supervisor |
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Dr. Mona M. Al-Sharif |
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Thesis Type |
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Master Thesis |
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Publishing Year |
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1439 AH
2018 AD |
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Co-Supervisor |
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Prof. Abdelbaset Buhmeida |
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Added Date |
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Thursday, May 31, 2018 |
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Researchers
| منى سالم الشيخ | Al-Shaikh, Mona Salem | Researcher | Master | |
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