Optimisation of polymerase chain reaction conditions for detection of mineralization markers in isolated odontoblasts from human teeth

ABSTRACT

The present study aimed to determine the best polymerase chain reaction (PCR) conditions for amplification of odontoblast markers; alkaline phosphatase (ALP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP) and osteopontin (OPN). Informed consent was obtained from the individuals prior to tooth extraction. RNA was extracted from odontoblasts obtained from extracted teeth using innuPREP RNA Mini kit (Analytik Jena, Germany). Five selected target factors in enhancing PCR primer concentration, extension time, number of cycles, annealing time, and annealing temperature were manipulated to yield the correct size of amplicons. One step reverse transcriptase PCR reactions were performed using MyTaq One-Step RT-PCR kit (Bioline, USA) with a C1000 Thermal Cycler (Bio-Rad, USA) in a 25 µL reaction, keeping the amount of 2 ng/µL RNA, 0.25 µL reverse transcriptase, 0.5 µL RiboSafe Rnase inhibitor and 1X MyTaq One-Step Mix, constant. The optimal conditions were determined to be 400nM of primers for DMP1 and DSPP, 200 nM for ALP and OPN; 30 seconds of extension time and 35 PCR cycles for all genes; 10 seconds of annealing time for ALP, DMP1 and DSPP, 7 seconds for OPN. The annealing temperature were 56.4°C for ALP, 58.6°C for DMP1, 52.7°C for DSPP, and 56.3°C for OPN, respectively. The optimized PCR protocols produced the correct size of odontoblast markers.