Mirvana qrt pcr mirna detection kit

RNA extraction was performed using TRIzol according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.). The synthesis of cDNAs was performed by reverse transcription reactions with 4 µg of total RNA using moloney murine leukemia virus reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.) with oligo dT (15 (link)) primers (Fermentas; Thermo Fisher Scientific, Inc.) as described by the manufacturer. The levels of miR-488 were quantified using the mirVana qRT-PCR miRNA detection kit (Ambion; Thermo Fisher Scientific, Inc.) in conjunction with RT-qPCR with SYBR Green. PCR reaction mixtures (final volume, 25 µl) were prepared, including 12.5 µl SYBR Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 2 µl cDNA, 300 nM each primer, and diethylpyrocarbonate-treated H₂O. The cycling conditions were 95°C for 5 min, followed by 38 cycles of 95°C for 15 sec, 58°C for 30 sec and 72°C for 30 sec. Following the cycle reaction, the quantification cycle (Cq) was determined and relative miR-488 levels were calculated based on the Cq values normalized to the levels of U6 in each sample (16 (link)). PCR was performed with the following primers: PHF8, forward 5′-GACATGTGCCAGGACTGGTTT-3′ and reverse 5′-CAGCAGCCTTCTCCTCTTCAA-3′; GAPDH, forward 5′-GCACCGTCAAGCTGAGAAC-3′ and reverse 5′-TGGTGAAGACGCCAGTGGA-3′.

Cellular miRNAs from hypoxia‐ or normoxia‐treated MC3T3‐E1 cells were extracted with an RNeasy Mini Kit (Qiagen, Valencia, CA), supplemented with RNase Inhibitor (Thermo Fisher Scientific, Rockford, IL), and then stored at − 80°C before use. miRNA samples for microarray analyses had a purity of 1.8 to 2.0 according to the absorbance at 260 nm/280 nm. miRNA samples were labeled with a Flash Tag Biotin HSR RNA Labeling kit (Affymetrix, Santa Clara, CA) and then hybridized with microRNA 4.0 Array (Affymetrix) under manufacturer protocols. Scan Array Express 1.0 (PerkinElmer, Waltham, MA) was utilized for scanning of hybridization signals, which were analyzed with Expression Console (Affymetrix). Each miRNA value was calculated by the log₂ transformation of normalized data. Quantitative real‐time polymerase chain reaction (qRT‐PCR) was undertaken to quantify deregulated miRNAs. qRT‐PCR was done with a mirVana qRT‐PCR miRNA Detection Kit (Thermo Fisher Scientific) according to the kit manual. The qRT‐PCR value was calculated by the 2−ΔΔCt method19 using U6 as the internal control.