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.
Background: Many smokers have undiagnosed chronic obstructive pulmonary disease (COPD), and yet screening for COPD is not recommended. Smokers who know that they have airflow limitation are more likely to quit smoking. This study aims to identify the prevalence and predictors of airflow limitation among smokers in primary care.
Methods: Current smokers ≥ 40 years old who were asymptomatic clinic attendees in a primary care setting were recruited consecutively for two months. We used a two-step strategy. Step 1: participants filled in a questionnaire. Step 2: Assessment of airflow limitation using a pocket spirometer. Multiple logistic regression was utilised to determine the best risk predictors for airflow limitation.
Results: Three hundred participants were recruited. Mean age was 58.35 (SD 10.30) years old and mean smoking history was 34.56 pack-years (SD 25.23). One in two smokers were found to have airflow limitation; the predictors were Indian ethnicity, prolonged smoking pack-year history and Lung Function Questionnaire score ≤ 18. Readiness to quit smoking and the awareness of COPD were low.
Conclusions: The high prevalence of airflow limitation and low readiness to quit smoking imply urgency with helping smokers to quit smoking. Identifying airflow limitation as an additional motivator for smoking cessation intervention may be considered. A two-step case-finding method is potentially feasible.
Study site: primary care clinic (outpatient clinic), Pulau Pinang, Malaysia