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

Kvantificering af telomerlængde ved hjælp af ikke-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.

Forfatter Spotlight: Optimering af ydeevneparametre for TAGGG telomerlængdeanalyse  

Optimering af ydeevneparametre for TAGGG Telomere Length Assay

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 og analyse

Begynd med at tilsætte 500 mikroliter lysisbuffer til den tidligere opnåede A2780-cellepellet, og bland forsigtigt ved hjælp af en skåret spids med en mindste åbningsdiameter på to millimeter. Tilsæt 20 mikrogram pr. milliliter frisklavet RNase og bland ved forsigtig invertering. Inkuber det ved 37 grader Celsius i 30 minutter.Ved afslutningen af inkubationen tilsættes proteinase K til en endelig koncentration på 100 mikrogram pr. Milliliter og inverteres forsigtigt 10 gange. Inkuber igen ved 55 grader Celsius i en time, inverter røret hvert 10. minut. Pipette 500 mikroliter phenol: chloroform: isoamylalkoholreagens ind i røret og vend forsigtigt røret ca. 20 gange.Centrifuger det ved 9.391 G i 15 minutter ved stuetemperatur. Det øvre vandige tyktflydende lag pipetteres ind i et nyt rør. Tilsæt et lige stort volumen chloroform og bland ved forsigtig inversion.Efter centrifugering af røret en gang samles det vandige lag. Tilsæt en passende mængde fem molært natriumchlorid i røret for at øge koncentrationen med 0,2 mol. Tilsæt to volumener 100% ethanol i røret og vend det forsigtigt 20 gange.Centrifuger ved 15.871 G i fem minutter ved stuetemperatur. Efter bortskaffelse af supernatanten tilsættes 500 mikroliter 70 % ethanol. Der centrifugeres igen ved samme tilstand, og supernatanten kasseres.Når pelleten er lufttørret, tilsættes 50 mikroliter sterilt nukleasefrit vand og lad det rehydrere. Bland derefter DNA'et ved pipettering ved hjælp af den afskårne spids. Brug UV-spektrofotometri til at måle koncentrationen af DNA.Efter fordøjelse af DNA'et adskilles det ved hjælp af 0,8% agarose. Agarosegelen nedsænkes i 0,25 molær saltsyreopløsning i 10 minutter ved stuetemperatur med forsigtig omrøring. Skyl derefter gelen to gange med destilleret vand.For at denaturere DNA'et nedsænkes gelen i en opløsning af natriumhydroxid og natriumchlorid. Skyl derefter gelen to gange med destilleret vand. For at neutralisere DNA'et som demonstreret nedsænkes gelen i en opløsning af 0,5 molær saltsyre og tre molære natriumchlorid ved pH syv.Ekstraktorgenomisk DNA viste god integritet, hvilket indikerer dets anvendelse til yderligere nedstrøms behandling af telomerrestriktionsfragmenter.

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 ...