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

Kvantifiering av telomerlängd med hjälp av icke-radioaktiv kemiluminescensdetektion

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.

Författare i fokus: Optimering av prestandaparametrar för TAGGG telomerlängdsanalys  

Optimering av prestandaparametrar för TAGGG-telomerlängdanalysen

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 Celler: Genomisk DNA-isolering och analys

Börja med att tillsätta 500 mikroliter av lysbufferten till den tidigare erhållna A2780-cellpelleten och blanda försiktigt med en skuren spets med en minsta öppningsdiameter på två millimeter. Tillsätt 20 mikrogram per milliliter nyberedd RNas och blanda genom försiktig inversion. Inkubera den vid 37 grader Celsius i 30 minuter.Vid slutet av inkubationen tillsätts proteinas K till en slutlig koncentration av 100 mikrogram per milliliter och invertera försiktigt 10 gånger. Inkubera igen vid 55 grader Celsius i en timme, vänd röret var 10: e minut. Pipettera 500 mikroliter fenol:kloroform:isoamylalkoholreagens i röret och vänd försiktigt röret cirka 20 gånger.Centrifugera den vid 9, 391 G i 15 minuter vid rumstemperatur. Pipettera det övre vattenhaltiga viskösa skiktet till ett nytt rör. Tillsätt en lika stor volym kloroform och blanda genom försiktig inversion.Efter centrifugering av röret en gång, samla vattenhaltigt skikt. Tillsätt en lämplig mängd fem molar natriumklorid i röret för att öka koncentrationen med 0,2 mol. Tillsätt två volymer 100% etanol i röret och vänd det försiktigt 20 gånger.Centrifugera vid 15 871 G i fem minuter vid rumstemperatur. Efter kassering av supernatanten, tillsätt 500 mikroliter 70% etanol. Centrifugera igen vid samma tillstånd och kassera sedan supernatanten.När pelleten är lufttorkad, tillsätt 50 mikroliter sterilt nukleasfritt vatten och låt det rehydrera. Blanda sedan DNA genom pipettering med hjälp av den skurna spetsen. Använd UV-spektrofotometri för att mäta koncentrationen av DNA.Efter att ha smält DNA, separera det med 0,8% agaros. Sänk ner agarosgelen i 0,25 molar saltsyralösning i 10 minuter vid rumstemperatur med mild omrörning. Skölj sedan gelén två gånger med destillerat vatten.För att denaturera DNA, nedsänk gelén i en lösning av natriumhydroxid och natriumklorid. Skölj sedan gelén två gånger med destillerat vatten. För att neutralisera DNA som visats, nedsänk gelén i en lösning av 0,5 molar saltsyra och tre molar natriumklorid vid pH sju.Extraktorns genomiska DNA visade god integritet, vilket indikerar dess användning för vidare nedströms bearbetning av telomerrestriktionsfragment.

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 ...