Validation of three reference genes for quantitative RT-PCR analyses in regenerating Lumbriculus variegatus

Abstract

Regeneration, the ability of organisms to replace body structures lost to injury, varies widely among and within animal taxa. The annelid Lumbriculus variegatus exhibits remarkable regeneration, growing both new head and tail regions from as few as three segments. Identifying orthologous genes upregulated early in regeneration in a variety of species may help pinpoint proteins that are essential for regeneration. Gene expression is frequently assayed with reverse transcription polymerase chain reaction (RT-PCR), a method that requires a validated reference gene. Therefore, we analyzed three common housekeeping genes to determine their suitability as reference genes during early L. variegatus regeneration. We found that all genes tested—18S ribosomal RNA, actin, and glyceraldehyde 3-phosphate dehydrogenase—showed stable RNA levels between intact worms and worms undergoing regeneration at 24 h post-amputation. Our validation of these reference genes will allow for more accurate RT-PCR studies in L. variegatus, and contribute to uncovering the mechanisms behind their remarkable regenerative abilities.