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

Cuantificación de la longitud de los telómeros mediante la detección de quimioluminiscencia no radiactiva

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.

Autor destacado: Optimización de los parámetros de rendimiento del ensayo de longitud de telómeros TAGGG  

Optimización de los parámetros de rendimiento del ensayo de longitud de telómeros TAGGG

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 Células Aislamiento y análisis de ADN genómico

Comience agregando 500 microlitros del tampón de lisis al pellet de celda A2780 obtenido previamente, y mezcle suavemente con una punta cortada con un diámetro de apertura mínimo de dos milímetros. Añadir 20 microgramos por mililitro de RNasa recién preparada y mezclar por inversión suave. Incubar a 37 grados centígrados durante 30 minutos.Al final de la incubación, agregue proteinasa K a una concentración final de 100 microgramos por mililitro e invierta suavemente 10 veces. Incubar de nuevo a 55 grados centígrados durante una hora invirtiendo el tubo cada 10 minutos. Pipetear 500 microlitros de fenol: cloroformo: reactivo de alcohol isoamílico en el tubo e invertir suavemente el tubo unas 20 veces.Centrifugar a 9, 391 G durante 15 minutos a temperatura ambiente. Pipetear la capa viscosa acuosa superior en un tubo nuevo. Añadir un volumen igual de cloroformo y mezclar mediante una inversión suave.Después de centrifugar el tubo una vez, recoger la capa acuosa. Agregue una cantidad apropiada de cloruro de sodio de cinco molares en el tubo para aumentar la concentración en 0.2 moles. Agregue dos volúmenes de etanol al 100% en el tubo e invierta suavemente 20 veces.Centrifugar a 15, 871 G durante cinco minutos a temperatura ambiente. Después de desechar el sobrenadante, agregue 500 microlitros de etanol al 70%. Centrifugar de nuevo en la misma condición y luego desechar el sobrenadante.Una vez que el pellet esté secado al aire, agregue 50 microlitros de agua estéril libre de nucleasas y deje que se rehidrate. Luego mezcle el ADN pipeteando usando la punta cortada. Utilice la espectrofotometría UV para medir la concentración de ADN.Después de digerir el ADN, sepárelo usando 0.8% de agarosa. Sumergir el gel de agarosa en solución de ácido clorhídrico molar 0,25 durante 10 minutos a temperatura ambiente con una agitación suave. Luego enjuague el gel dos veces con agua destilada.Para desnaturalizar el ADN, sumerja el gel en una solución de hidróxido de sodio y cloruro de sodio. Luego enjuague el gel dos veces con agua destilada. Para neutralizar el ADN como se demuestra, sumerja el gel en una solución de ácido clorhídrico molar 0,5 y cloruro de sodio tres molares a pH siete.El ADN genómico del extractor mostró una buena integridad, lo que indica su uso para el procesamiento posterior de fragmentos de restricción de telómeros.

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 ...