Scholar Hub/Chủ đề/#rt pcr/
RT-PCR (Reverse Transcription Polymerase Chain Reaction) là một phương pháp di truyền phân tử được sử dụng để nhân đôi và phân tích một đoạn cụ thể của RNA trong mẫu. Phương pháp này thường được sử dụng để phát hiện và xác định sự hiện diện của vi rút hoặc gen cụ thể trong mẫu sinh phẩm. RT-PCR được sử dụng rộng rãi trong lĩnh vực y học, chẩn đoán bệnh, nghiên cứu di truyền và nhiều lĩnh vực khác.
RT-PCR là một kỹ thuật phân tử sinh học được sử dụng để phát hiện và nhân bản các đoạn RNA cụ thể trong mẫu. Phương pháp này kết hợp giữa một bước đảo ngược transcripsion (reverse transcription) để chuyển đổi RNA thành DNA một một tên gọi khác là cDNA (complementary DNA) và sau đó sử dụng phương pháp polymerase chain reaction (PCR) để nhân bản DNA này.
Quá trình RT-PCR bao gồm các bước chính như sau:
1. Reverse Transcription (RT): Trước tiên, enzyme đảo ngược transcripsion (reverse transcriptase) được sử dụng để chuyển đổi RNA thành cDNA. Một đoạn oligonucleotide ngược (primer) được thêm vào để bắt đầu quá trình transcripsion. RT xảy ra trong một ống nhiệt độ đã được điều chỉnh để cung cấp các điều kiện lý tưởng cho reverse transcriptase hoạt động.
2. PCR: Sau khi cDNA được tạo ra, phương pháp PCR được sử dụng để nhân bản DNA nhằm tăng lượng đoạn DNA mong muốn lên. PCR bao gồm các chu kỳ nhiệt đới kéo dài, mỗi chu kỳ bao gồm các bước gia nhiệt để tách các một đúng nhân đôi (denaturation), một bước gia nhiệt để các primer được gắn vào mỗi sợi mẫu (annealing), và một bước gia nhiệt để enzyme polymerase sao chép đoạn DNA (extension). Quá trình tổng cộng này được lặp lại nhiều lần để tạo ra hàng triệu bản sao của DNA mục tiêu.
3. Phân tích kết quả: Khi quá trình PCR hoàn tất, sản phẩm nhân bản có thể được phân tích bằng cách sử dụng các phương pháp như gel điện phoresis, qPCR (quantitative PCR), sequencing, hoặc sử dụng các công cụ phân tích khác.
RT-PCR là một công cụ mạnh mẽ trong nghiên cứu gen và di truyền, y tế và chẩn đoán bệnh. Nó được sử dụng để phát hiện và xác định sự hiện diện của RNA từ một loại vi rút cụ thể, như vi rút SARS-CoV-2 gây ra COVID-19, hoặc để theo dõi mức độ biểu hiện gen trong mẫu sinh phẩm.
Generation of an Immortalized Erythroid Progenitor Cell Line Directly from Peripheral Blood Mononuclear Cells without Using Mobilized CD34+ Cells Blood - Tập 132 - Trang 1032 - 2018
Abhirup Bagchi, Aneesha Nath, Vignesh Rajendiran, Madhavi Maddali, Ekta Jajodia, Mohankumar Kumarasamypet Murugesan, Yukio Nakamura, Alok Srivastava, Shaji Ramachandran Velayudhan
Abstract
A reliable stable human erythroid progenitor cell line that can differentiate to the later stages of erythropoiesis is an important cellular model for studying molecular mechanisms of human erythropoiesis in physiological and pathological situations. An erythroid progenitor cell line (HUDEP) was derived from cord blood haematopoietic stem cells (HSCs) by doxycycline inducible expression of HPV E6/E7 gene (Kurita et al., 2013). This cell line could be differentiated further to terminally differentiated red cells, and it has been extensively used for studying transcriptional regulation of human erythropoiesis. Using the same strategy, immortalized erythroid progenitors could also be generated from adult HSCs (Trakarnsanga et al, 2017). However, generation of immortalized erythroid cells from patients using this protocol is challenging as obtaining sufficient number of adult HSCs requires mobilization of HSCs using GCSF. Peripheral blood mononuclear cells (PBMNCs) contain a small number of erythroid progenitors, which can be expanded and differentiated in culture. Till date, there are no reports on the generation of immortalized erythroid progenitors directly from PBMNCs. In this study, we established a protocol for the generation of immortalized erythroid progenitors from PBMNCs of a normal donor. The PBMNCs isolated from 10ml of blood from a normal donor were cultured for 24 hours in the primary erythroid expansion medium as described earlier (Trakarnsanga et al, 2017). These cells were then transduced with lentiviruses to express HPV E6/E7 gene and a fluorescent protein hKO1. After 3 days, the cells were cultured in a serum free medium containing the cytokines (stem cell factor and erythropoietin) and dexamethasone in the presence of doxycycline for 15 days. The cells that expressed hKO1 were sorted by FACS, and they were cultured in the same medium till the immortalization was complete. We continuously monitored the cells for the kinetics in the expression of erythroid cell surface markers, CD36, CD71 and CD235a, till >95% of the cells expressed all these markers. On day 50, all the cells expressed high levels of the erythroid markers and the cell morphology analysis using Giemsa staining showed that 65% of the cells were pronormoblasts, 22% were basophilic normoblasts and the rest of the cells were at the later stages of differentiation. To evaluate the differentiation potential of these cells, the cells were cultured using the media and conditions described by Hawksworth et al, 2018. After the removal of doxycycline from the culture medium, the cells showed haemoglobinization and the morphology analysis showed that 10% of the cells were in the polychromatic stage and 88% of the cells were in the orthochromatic stage, suggesting robust erythroid differentiation of the immortalized erythroid progenitors with the suitable cell culture conditions. These data showed that immortalized erythroid progenitors with differentiation potential could be generated directly from peripheral blood without using mobilized haematopoietic stem cells. This protocol is suitable for the generation of immortalized erythroid cells from the patients with rare red cell genetic disorders for studying disease mechanisms.
Disclosures
No relevant conflicts of interest to declare.
Is CuO a spin fluid? Journal of Magnetism and Magnetic Materials - Tập 89 - Trang L277-L283 - 1990
K. Muraleedharan
A time-dependent phenomenological model for cell mechano-sensing Biomechanics and Modeling in Mechanobiology - Tập 13 - Trang 451-462 - 2013
Carlos Borau, Roger D. Kamm, José Manuel García-Aznar
Adherent cells normally apply forces as a generic means of sensing and responding to the mechanical nature of their surrounding environment. How these forces vary as a function of the extracellular rigidity is critical to understanding the regulatory functions that drive important phenomena such as wound healing or muscle contraction. In recognition of this fact, experiments have been conducted to understand cell rigidity-sensing properties under known conditions of the extracellular environment, opening new possibilities for modeling this active behavior. In this work, we provide a physics-based constitutive model taking into account the main structural components of the cell to reproduce its most significant contractile properties such as the traction forces exerted as a function of time and the extracellular stiffness. This model shows how the interplay between the time-dependent response of the acto-myosin contractile system and the elastic response of the cell components determines the mechano-sensing behavior of single cells.
Generalization of EDF and LLF: Identifying All Optimal Online Algorithms for Minimizing Maximum Lateness Algorithmica - Tập 50 - Trang 312-328 - 2007
Patchrawat “Patch” Uthaisombut
It is well known that the Earliest-Deadline-First (EDF) and the Least-Laxity-First (LLF) algorithms are optimal algorithms for the problem of preemptively scheduling jobs that arrive over time on a single machine to minimize the maximum lateness (1|r
j
,pmtn|L
max ). It was not previously known what other online algorithms are optimal for this problem. As this problem is fundamental in machine scheduling, it deserves a thorough investigation. In this paper, the concept of compound laxity is introduced, and a complete characterization of all optimal online algorithms for this problem is derived.
Sensor proteins JBIC Journal of Biological Inorganic Chemistry - Tập 14 - Trang 169-172 - 2009
Studies in Fractionated Montmorillonite Suspensions Clays and Clay Minerals - Tập 11 - Trang 282-294 - 1962
Necmettin Mungan, Frank W. Jessen
Four natural bentonites, from Texas, Wyoming, Mexico, and Puerto Rico, have been fractionated by use of a super-centrifuge. The fractions have been investigated for base exchange capacity, apparent density, X-ray diffraction character and gel strengths of their suspensions. Based on (001) spacings from X-ray diffraction and cation composition from ammonium acetate base exchange data, it was found that upon fractionation, clays of heterogeneous cation composition divide into two groups. Fractions larger than 0.150 micron contain the divalent cations Ca2+-Mg2+, while the finer fractions contain the monovalent cation Na+. Based on indirect evidence provided by X-ray diffraction, particle size fractionation, and gel strength, and correlation of the data with the literature, it is concluded that Na+ clays exist as unit layers in suspensions while Ca2+-Mg2+ particles are aggregates of a few layers. Base exchange capacity and density were found to be independent of particle size. A new structure is postulated and proposed for aqueous montmorillonite gels. This structure, descriptively called “hinge,” comprises clay particles oriented randomly to form a network in which each particle is hinged to other particles through sharing of bound particle-water.
