Lc480 qpcr machine

First strand synthesis was executed using Universal cDNA synthesis kit (Exiqon) according to the manufacturer's instructions. Owing to its superior sensitivity, the SYBR Green master mix and specific-miRNA (miR-17 and miR-20a) LNA primers (Exiqon) were used according to the manufacturer's instructions for microarray validation and detection of specific miRNAs. Detection was carried out using the LC480 qPCR machine (Roche) according to the manufacturer's guidelines, followed by melting curve analysis at the end of the run.

Total RNA was isolated with the Invitrogen™ TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Total RNA (1.0 μg) was reverse transcribed using the M-MLV reverse transcriptase (Promega Corp., Madison, WI, USA) to synthesize complementary DNA. The primers used for qPCR were as follows: TLR-4 forward (F), 5′-GCTTTCACCTCT GCCTTCAC-3′, and reverse (R), 5′-CCAACGGCTCTG AATAAAGTG-3′; 3-phosphate dehydrogenase (GAPDH) F, 5′-TCACCACCATGGAGAAGGC-3′, and R, 5′-GCTAAG CAGTTGGTGGTGCA-3′. qPCR was performed with the following cycling parameters: 40 cycles of denaturation at 94°C for 20 sec, annealing at 62°C for 30 sec, and extension at 72°C for 30 sec. The SYBR-Green qPCR Master Mix 2 kit (Takara Bio Inc., Otsu, Japan) was used in all samples, and the reactions were carried out in a 20 μl final reaction volume, using an LC480 qPCR machine (Roche, Basel, Switzerland). The mRNA expression levels of target genes were calculated based on standard curve analysis with the absolute quantification method, and were expressed relative to the level of GAPDH, a housekeeping gene used as an endogenous control (12 (link)).

First, cDNA of genes was generated by transcriptor universal cDNA master kit (Roche, Penzberg Germany) and cDNA of miRNAs by miRCURY LNA™ universal cDNA synthesis kit (Exiqon, Vedbaek Denmark) according to the manufacturer's instructions.
Gene expression was measured with the SYBR green I master (Roche, Penzberg Germany) and gene-specific primers. For miRNAs detection, the SYBR Green master mix and specific-miRNA LNA primers were used according to the manufacturer's instructions (Exiqon, Vedbaek Denmark). The real-time PCR (qPCR) reactions were normalized to GAPDH or SNORD48 endogenous control respectively.
Detection was carried out using the LC480 qPCR machine (Roche, Penzberg Germany) according to the manufacturer's guidelines, followed by melting curve analysis at the end of the run.