Name: A2780 Cells Genomic DNA Isolation and Analysis
Uploaded: 2023-04-21T13:20:00
Description: Watch this Scientific Journal Video about A2780 Cells Genomic DNA Isolation and Analysis at JoVE.com

The video outlines a detailed protocol for extracting genomic DNA from A2780 cells. It covers the use of lysis buffer, RNase, and proteinase K, followed by phenol-chloroform extraction and precipitation with sodium chloride and ethanol. The process ensures high-quality DNA suitable for analysis of telomere restriction fragments.

a2780 cells genomic dna isolation and analysis

Non-Radioactive Chemiluminescence Detection을 사용한 텔로미어 길이 정량화

Telomeres are the repetitive DNA sequences present at the end of chromosomes. They have tandem repeats of TTAGGG and maintain genome integrity by protecting the chromosome from both fraying and the end replication problem, which means that part of the 3&#39; overhang is unable to be replicated by DNA polymerase1,2. Short telomeres lead to chromosomal abnormalities in cells, due to which cells become permanently arrested in a stage called replicative senescence3. Short telomeres also cause a host of other problems, such as mitochondria dysfunction4,5 and cell dysfunction.
DNA telomeric repeats are lost as and when the cell divides, with an average loss of 25 to 200 bp per year6, resulting in cellular senescence after a certain number of divisions6. Aging is associated with a higher frequency of comorbidities, which is marked by a shortening in telomere length7. Telomere restriction fragment (TRF) analysis, as described by Mender, is a very expensive method8. Because of this, it is not implemented while quantifying telomere length in most studies.
Presently, the majority of epidemiological studies employ quantitative polymerase chain reaction (qPCR)-based measurements of telomere length. However, the qPCR-based method is a relative measurement method, as it measures the ratio between telomeres and single-copy gene amplification products, and not absolute telomere length. Telomere length measurement using the TRF protocol is the gold standard method, as it can measure telomere length distribution in the sample and measurements can be expressed in absolute values in kilobases (kb). However, its use is limited because it is cumbersome, labor-intensive, and costly. Here, we present an optimized protocol for telomere length measurement using chemiluminescence-based TRFs.
TRF analysis includes seven major steps: 1) culturing of cells for genomic DNA extraction, 2) genomic DNA extraction using the phenol:chloroform:isoamylalcohol (P:C:I) method, 3) restriction digestion of genomic DNA, 4) agarose gel electrophoresis, 5) Southern blotting of the restriction digestion DNA fragment, 6) hybridization and detection via chemiluminescence-the immobilized telomere probe is visualized by a highly sensitive chemiluminescent substrate for alkaline phosphatase, disodium 2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2&#8242;-(5-chlorotricyclo[3.3.1.13.7]decan])-4-yl]-1-phenyl phosphate (CDP-Star)-and 7) analysis for obtaining mean telomere length and range information from these telomeric smears.

저자 스포트라이트: TAGGG 텔로미어 길이 분석의 성능 파라미터 최적화  

TAGGG 텔로미어 길이 분석의 성능 매개변수 최적화

The scope of our research involves measuring telomere length differences across various study groups. The questions we are trying to answer revolve around diseases of physiological conditions and how they may affect telomere length and vice versa. The technology is currently used to advance research in the field of telomere length biology are nanopore sequencing and more recently CRISPR Cas based measurement.Current experimental challenges are accurately measuring absolute telomere length and the changes in it. The same sample could give varying results when different methods of measuring telomere length are used. The research gap we address is the lack of a standard method to measure telomere length.Telomere restriction fragment analysis or TRF, is still the standard against which all new techniques are evaluated. Also, our optimized protocols helps in processing a higher number of samples with limited resources. Our findings will help advance research in the field of telomere length biologic by providing a reliable and relatively economical way to compare newer methods to the gold standard, which is TRF.Our laboratory will focus on the effects of small molecule inhibitors on telomere length. We will also study the impact that lifestyle diseases and telomere length have on on each other.

Watch this Scientific Journal Video about A2780 Cells Genomic DNA Isolation and Analysis at JoVE.com

A2780 Cells Genomic DNA Isolation and Analysis  

A2780 세포 게놈 DNA 분리 및 분석

이전에 얻은 A2780 세포 펠릿에 500 마이크로 리터의 용해 완충액을 첨가하고 최소 개구 직경이 2 밀리미터 인 절단 팁을 사용하여 부드럽게 혼합합니다. 갓 준비한 RNase를 밀리리터당 20마이크로그램 첨가하고 부드럽게 반전시켜 혼합합니다. 섭씨 37도에서 30 분 동안 배양하십시오.배양이 끝나면 proteinase K를 밀리리터 당 100 마이크로 그램의 최종 농도에 첨가하고 부드럽게 10 회 반전시킵니다. 섭씨 55도에서 1시간 동안 다시 배양하여 10분마다 튜브를 뒤집습니다. 페놀:클로로포름:이소아밀 알코올 시약 500마이크로리터를 튜브에 넣고 튜브를 약 20회 부드럽게 뒤집습니다.실온에서 15 분 동안 9, 391 G에서 원심 분리한다. 상부 수성 점성 층을 새 튜브에 피펫팅합니다. 같은 양의 클로로포름을 첨가하고 부드러운 반전으로 혼합하십시오.튜브를 한 번 원심분리한 후 수층을 수집합니다. 튜브에 적절한 양의 5몰 염화나트륨을 첨가하여 농도를 0.2몰 증가시킵니다. 튜브에 100% 에탄올 2개를 넣고 부드럽게 20회 뒤집습니다.실온에서 5 분 동안 15, 871 G에서 원심 분리한다. 상청액을 버린 후 500 마이크로 리터의 70 % 에탄올을 첨가하십시오. 동일한 조건에서 다시 원심분리한 다음 상층액을 버립니다.펠릿이 공기 건조되면, 50 마이크로리터의 멸균 뉴클레아제가 없는 물을 추가하고 재수화시킵니다. 그런 다음 절단된 팁을 사용하여 피펫팅하여 DNA를 혼합합니다. UV 분광 광도법을 사용하여 DNA의 농도를 측정합니다.DNA를 소화한 후 0.8% agarose를 사용하여 분리합니다. 아가로스 겔을 0.25 몰 염산 용액에 담가두고, 실온에서 10분 동안 부드럽게 교반한다. 그런 다음 증류수로 젤을 두 번 헹굽니다.DNA를 변성시키려면 겔을 수산화나트륨과 염화나트륨 용액에 담그십시오. 그런 다음 증류수로 젤을 두 번 헹굽니다. 입증된 바와 같이 DNA를 중화하기 위해, 겔을 pH 7에서 0.5몰 염산 및 3몰 염화나트륨의 용액에 담근다.추출기 게놈 DNA는 텔로미어 제한 단편의 추가 다운스트림 처리에 사용됨을 나타내는 우수한 무결성을 보여주었습니다.

Research

JoVE Journal

Cancer Research

A2780 Cells Genomic DNA Isolation and Analysis

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...

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연구

암 연구학

Southern Blotting, Hybridization, and Chemiluminescence Detection of Genomic DNA Isolated from A2780 Cells

A2780 세포에서 분리된 게놈 DNA의 Southern Blotting, Hybridization, and Chemiluminescence Detection of Genomic DNA Isolation (A2780 세포에서 분리된 게놈 DNA의 Southern Blotting, Hybridization, and Chemiluminescence Detection)