Validation of the use of 32P-orthophosphate as a probe for monitoring DNA replication in pancreatic islet cells.
This study aims at validating the use of 32P-orthophosphate as a probe for monitoring DNA replication instead of [3H]-thymidine where the specific activity of the latter is affected by perturbation of the cellular environment. It was found that to obtain a clean DNA fraction for determining the incorporation of the label, protein must be removed by the use of Proteinase K, and RNA by alkaline hydrolysis. Alkaline hydrolysis of RNA was found superior to RNAse digestion in not leaving behind large RNA fragments that may contaminate the DNA fraction.
D000818:Animals; D002478:Cells, Cultured; D004261:DNA Replication; D005333:Fetus; D007515:Islets of Langerhans / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D011865:Radioisotope Dilution Technique; D051381:Rats; D013936:Thymidine / Q000378:metabolism; D014316:Tritium
D010710:Phosphates; D010761:Phosphorus Radioisotopes; D014316:Tritium; D013936:Thymidine