Mirvana paris extraction kit

Total RNA was extracted from 300 μL of saliva supernatant using the mirVana PARIS extraction kit (Ambion). On-column DNase treatment (Qiagen) was used to remove contaminating DNA during RNA extraction. Total RNA (3 ng) was converted to complementary DNA using the TaqMan miRNA Reverse Transcription Kit (Applied Biosystems). Two different sets of stem-loop RT primers (human pool A and human pool B) were used (Megaplex RT primers, Applied Biosystems). After reverse transcription, the RT product was preamplified using TaqMan PreAmp Master Mix (Applied Biosystems) and Megaplex PreAmp primers (Applied Biosystems). The preamp product was not diluted before miRNA quantification. The TaqMan Human miRNA array set version 3.0 (Applied Biosystems) and TaqMan Universal PCR Master Mix, no AmpErase uracil N-glycosylase were used for miRNA quantification. All reactions were performed on a 7900HT Fast Real-Time PCR System containing a special cardholder (Applied Biosystems). Data were analyzed using RQ Manager software version 1.2 and DataAssist software version 3.0 (Applied Biosystems). Similarly, the ∆Cq value was computed using RNA polymerase III transcribed U6 small nuclear RNA as the reference gene.