Taqman fast pcr kit

To assess microRNA and gene mRNA expression levels, the TaqMan Fast-PCR kit (Applied Biosystems, Foster City, CA, USA) was utilized according to the manufacturer’s instructions, followed by detection with the 7900HT Sequence Detection System (Applied Biosystems). All reactions were performed in technical triplicate. Simultaneous quantification of RNU6 and GAPDH was used as reference for miRNA and gene quantification, respectively. The comparative cycle threshold (Ct) method for relative quantification of gene and miRNA expression (User Bulletin #2; Applied Biosystems) was employed.

RNA was purified with the “RNA clean-up and concentration” micro kit (Norgen) according to the manufacturer's instructions, after phenol/guanidine-based extraction. For RNA extraction from EVs, Ath-miR159a and cel-miR-248 synthetic oligos were added to each sample to normalize the results of quantitative RT-PCR. Quantitative real-time PCRs were performed with the TaqMan Fast-PCR kit (Applied Biosystems) according to the manufacturer's instructions, using the appropriate TaqMan probes for miRNA quantification, followed by detection with the 7900HT Sequence Detection System (Applied Biosystems). All reactions were performed in triplicate. Simultaneous quantification of GAPDH was used as reference for intracellular miRNA quantification. Simultaneous quantification of RNU48 was used as reference for exosome-related miRNA quantification. The comparative cycle threshold (Ct) method for relative quantification of gene and miRNA expression (User Bulletin #2; Applied Biosystems) was used to determine miRNA expression levels.

Total RNA was extracted using TRIzol (Invitrogen) according to manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) were performed using the TaqMan Fast-PCR kit (Applied Biosystems) according to the manufacturer's instructions, using the appropriate TaqMan probes for miRNA and gene quantification, followed by detection with the 7900HT Sequence Detection System (Applied Biosystems). All reactions were performed in triplicate. Simultaneous quantification of RNU44 was used as reference for miRNA quantification. Simultaneous quantification of GAPDH mRNAs was used as reference for gene quantification. The comparative cycle threshold (Ct) method for relative quantification of gene and miRNA expression (User Bulletin #2; Applied Biosystems) was used to determine miRNA and gene expression levels.