Quantification of TREM-1 and TREM-2 Expression in Tendon Tissue

Total RNA (1μg) from tendon tissue was reverse transcribed to cDNA using PROMEGA cDNA synthesis kit according to the manufacturer’s protocol. The RNA and oligo-dT primers were primed prior to the reaction. Reaction mixture (MgCl₂, reaction buffer, dNTP mix, RNase inhibitor and reverse transcriptase) was prepared for 20μl reaction volume and cDNA synthesis was carried out in Bio-Rad T100 thermal cycler. Gene expression levels of TREM-1 and TREM-2 were quantified by the real-time PCR system (Bio-Rad T1000 thermal cycler). The 8μl cDNA (1:1 dilution) was mixed with 10μl SYBR Green PCR master mix (PROMEGA) and corresponding forward and reverse primers (10pM/μl) (Table 1) for real-time PCR amplification. The 18s rRNA was used as the housekeeping gene. The reaction conditions were 95°C for 10 minutes, 40 cycles at 95°C for15s, and 60°C for 1 min. The mRNA expression of TREM-1 and TREM-2 was normalized with the expression level of 18s rRNA. Four independent experiments were carried out for each sample and average of Cq values was taken. Results are represented as fold-change of TREM-1 and TREM-2 mRNA levels between patients and the reference gene [41 (link)].