Mircury lna universal cdna synthesis kit 2

The miRCURY LNA™ Universal cDNA Synthesis Kit II (Exiqon, Vedbaek, Denmark) was used to prepare cDNA. RT-PCR was carried out using SYBR Green PCR Master Mix (YectaTajhizAzma, Iran) and miRNA-LNA™ PCR primers at 200 nM concentration to measure miR-1 expression in MDA-MB-231 and MCF-7 cells (Table 1). The SYBR Green RT-PCR conditions were as follows: 10 minutes at 95°C, 40 cycles 10 seconds at 95°C (denaturation), and 30 seconds at 60°C (annealing). The nontemplate control was prepared using nuclease-free water. The relative miR-1 expression of untreated and HCF-treated breast cancer cells was analyzed using the Ct (2–^ΔΔCt) formula. The internal control for this analysis was the expression level of the U6 snRNA. The values of the treated breast cancer cell lines were compared to those of the untreated ones.

Total RNA was isolated from FFPE tissue samples using miRCURY RNA isolation FFPE (Exiqon Inc., Vedbaek, Denmark). For cell RNA isolation we used the miRNeasy Mini Kit (Qiagen). RNA concentration and purity were evaluated using a spectrophotometer (Nanodrop 2000, Thermo Scientific, Madison, WI, USA). Total RNA was reverse transcribed with the miRCURY LNA Universal cDNA Synthesis Kit II (Exiqon Inc., Vedbaek, Denmark), according to the manufacturer’s instructions.

RNA for profiling of miRNA was isolated from FFPE tissue using the RecoverAll™ total nucleic acid isolation kit (Applied Biosystems, Carlsbad, California, USA). RNA concentrations were quantified using the NanoDrop Spectrophotometer (Nanodrop Technologies, Montchanin, New Castle, Delaware, USA). Afterwards, reverse transcription (RT) using amounts of 20 ng of total RNA by applying the miRCURY LNA™ Universal cDNA Synthesis Kit II (Exiqon, Vedbaek, Denmark), containing synthetic RNA Spike Ins, was performed. 5 μl of the RT products was combined with the PCR master mix and nuclease-free water from the miRCURY LNA™ ExiLENT SYBR® Green master mix (Exiqon, Vedbaek, Denmark). After that, 10 μl of the PCR Master mix-cDNA mix was added to each 384-well plate of the miRCURY LNA™ Universal RT miR Ready-to-Use PCR, Cancer focus panel, V4 (Exiqon, Vedbaek, Denmark). Finally, qPCR was performed by using the LightCycler® 480 instrument (Roche molecular systems Inc., Mannheim, Germany). All reactions were carried out according to the manufacturer's instructions. Initial data analysis was executed by using the LightCycler® 480 Software (Roche molecular systems Inc., Mannheim, Germany) to obtain raw Ct values. Ct values were used to determine the amount of miRNA in a sample (both parameters showing an inverse correlation).

cDNA was synthesized using a miRCURY LNA Universal cDNA Synthesis Kit II (Exiqon) starting from 6 μl of total RNA with the addition of 1 μl of UniSp6 and 1 μl of Cel-miR-39 as exogenous miRNA spiked-in control. The RT product was used as a template in the qPCR assays in Ready-to-use Human Cancer Focus microRNA PCR Panel V4 (Supplementary Table S1, Exiqon) and ExiLENT SYBR Green master mix (Exiqon). ROX (Invitrogen by Life Technologies, Carlsbad, CA, United States) was added to the master mix as the passive reference dye. The arrays were run in an ABI7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, United States). The qPCR cycling conditions were 95°C for 10 min and 40 cycles (95°C for 10 s and 60°C for 1 min).

Isolation of total miRNA was performed using the miRCURY RNA Isolation Kit (300110; Exiqon, Vedbaek, Denmark), with some modifications. Briefly, oocytes and embryos (20 per group) were lysed in 350 μl lysis buffer, mixed with 200 μl of 100 % ethanol and pipetted directly onto an RNA-binding column. After washing, miRNA was eluted using 50 μl elution buffer followed by a second elution with 50 μl of RNAse-free water. The eluent was concentrated using the RNeasy MinElute Cleanup Kit (74204, Qiagen, Valencia, CA, USA). Reverse transcription (RT) was performed using a miRCURY LNA, Universal cDNA Synthesis Kit II, (203301, Exiqon) in a total volume of 20 μl made up of 10 μl sample RNA, 4 μl 5x buffer, 2 μl RNAse-free water, 2 μl RNA spike (UniSp6) and 2 μl enzyme mix. The mixture was incubated for 1 h at 42 °C, followed by 5 min at 80 °C before storage at −20 °C.

Total RNA was isolated from LV tissue of sham, post-MI, hypo-, and hyperthyroid mice using mirVana PARIS kit for miRNA (Ambion, Foster City, CA, USA) or TriPure for mRNA (Roche, Basel, Switzerland) and treated with DNaseI (Qiagen, Venlo, The Netherlands) to remove remnants of genomic DNA, followed by reverse transcription reaction with either Cloned AMV First Strand synthesis kit (Invitrogen, Carlsbad, CA, USA) or with miRCURY LNA Universal cDNA synthesis kit II (Exiqon, Vedbæk, Denmark). Expression levels of atrial natriuretic factor (Anf), MHCα (Myh6), MHCβ (Myh7), and Dio3 mRNA were determined by qPCR using specific primers (Table S1 in Supplementary Material) and standard cycle parameters on an Applied Biosystems model 7500 (Applied Biosystems, Foster City, CA, USA) with hypoxanthine-guanine-phosphoribosyl-transferase (Hprt) as correction factor. Expression levels of miR-214-3p were analyzed using miRCURY LNA miRNA primers and normalized against U6 snRNA (Exiqon, Vedbæk, Denmark).

In order to evaluate miRNA expression levels and profiles of AHE, CHE groups, and HEV-negative control group, analysis of 180 miRNA species was performed using miRCURY LNA Universal cDNA Synthesis Kit II (Exiqon, Vedbaek, Denmark) for reverse transcription, ExiLENT SYBR Green master mix (Exiqon, Vedbaek, Denmark) for quantitative PCR (qPCR) amplification and Serum/Plasma Focus qPCR Panels V4.RO (Exiqon, Vedbaek, Denmark) according to the manufacturer´s instructions. As internal amplification controls synthetic miRNAs UniSp2, 4 and 5 were added as spike-ins before extraction and UniSp6 before cDNA synthesis (Exiqon, Vedbaek, Denmark). Data analysis was performed with GenEx qPCR analysis software (Exiqon, Vedbaek, Denmark). Raw cycle threshold (Ct) values were inter-plate calibrated using UniSp3. To identify endogenous reference miRNAs and regulated miRNAs for expression analyses during HEV infection, global mean normalization was performed. In brief, fold changes (FC) of miRNA expression in AHE and CHE patients compared to controls were calculated using the 2^−ΔΔCt method. Four regulated miRNAs were selected for further investigation based on previous studies^{24 (link)–30 (link)}. The three most stably expressed miRNAs were selected as endogenous reference genes.