Detection of Apoptotic DNA Ladder in Pig Leukocytes and its Precision Using LM-PCR (Ligation Mediated Polymerase Chain Reaction)

Abstract

Apoptosis as a way of programmed cell death represents an extremely important phenomenon in development and everyday maintenance of homeostasis in multicellular organisms. Regarding the keen interest in programmed cell death in the last ten years, more and more assays and kits are emerging to prove apoptosis. However, their detectability and reliability have been often discussed. Apoptotic DNA changes in pig leukocytes as a consequence of phagocytosis were investigated by two methods available for qualitative apoptosis evaluation in cell populations. Traditionally used and quick screening of apoptotic DNA fragmentation by agarose gel electrophoresis showed negative results or a non-typical apoptotic ladder. The new approach using LM-PCR technique was tested to make those results more precise and to find out if a typical DNA ladder can be obtained. Apoptotic cells were quantified using TUNEL test. LM-PCR technique has been shown not only as a method for result precision in the case of negative findings by DNA ladder assay but also as an effective assay in case of non-typical apoptotic DNA ladder appearance. Moreover, LM-PCR detected an apoptotic ladder even in populations with less than 1% of apoptotic cells.