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

Kvantifisering av telomerlengde ved bruk av ikke-radioaktiv kjemiluminescensdeteksjon

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.

Forfatter i søkelyset: Optimalisering av ytelsesparametere for TAGGG telomerlengdeanalyse  

Optimalisering av ytelsesparametere for TAGGG-telomerlengdeanalysen

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-isolasjon og analyse

Begynn med å tilsette 500 mikroliter av lysisbufferen til den tidligere oppnådde A2780-cellepelleten, og bland forsiktig med en kuttspiss med en minimum åpningsdiameter på to millimeter. Tilsett 20 mikrogram per milliliter nytilberedt RNase og bland med mild inversjon. Inkuber den ved 37 grader Celsius i 30 minutter.På slutten av inkubasjonen, tilsett proteinase K til en endelig konsentrasjon på 100 mikrogram per milliliter og vend forsiktig 10 ganger. Inkuber igjen ved 55 grader Celsius i en time, og inverter røret hvert 10. minutt. Pipette 500 mikroliter fenol:kloroform:isoamylalkoholreagens inn i røret og vend forsiktig røret ca. 20 ganger.Sentrifuger den ved 9.391 g i 15 minutter ved romtemperatur. Pipett det øvre vandige viskøse laget inn i et nytt rør. Tilsett et likt volum kloroform og bland med forsiktig inversjon.Etter å ha sentrifugert røret en gang, samle det vandige laget. Legg til en passende mengde fem molare natriumklorid i røret for å øke konsentrasjonen med 0,2 mol. Tilsett to volumer 100% etanol i røret og snu det forsiktig 20 ganger.Sentrifuge ved 15.871 G i fem minutter ved romtemperatur. Etter å ha kastet supernatanten, tilsett 500 mikroliter 70% etanol. Sentrifuger igjen i samme tilstand og kast deretter supernatanten.Når pelleten er lufttørket, tilsett 50 mikroliter sterilt nukleasefritt vann og la det rehydrere. Bland deretter DNA-et ved å pipettere med kuttspissen. Bruk UV-spektrofotometri for å måle konsentrasjonen av DNA.Etter å ha fordøyd DNA, skille det ved hjelp av 0,8% agarose. Senk agarosegelen i 0,25 molar saltsyreoppløsning i 10 minutter ved romtemperatur med forsiktig omrøring. Skyll deretter gelen to ganger med destillert vann.For å denaturere DNA, senk gelen i en løsning av natriumhydroksid og natriumklorid. Skyll deretter gelen to ganger med destillert vann. For å nøytralisere DNA som demonstrert, senk gelen i en løsning av 0,5 molær saltsyre og tre molare natriumklorid ved pH syv.Ekstraktorgenomisk DNA viste god integritet, noe som indikerer bruk for videre nedstrøms prosessering av telomerrestriksjonsfragmenter.

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 ...