Mircury lna rt kit

A fixed volume of 5 ng/μL of total RNA was used for the RT reaction. First‐strand cDNA synthesis was performed using the miRCURY® LNA® RT Kit according to the manufacturers' protocol (Exiqon). cDNA was diluted (1:80) in nuclease‐free H₂O and stored at 4 °C, and cDNA stocks were stored at −20 °C. For real‐time PCR amplification, each reaction contained 5 μL of ExiLENT SYBR® Green master mix (Exiqon), 1 μL of miRCURY LNA PCR primer mix (Exiqon), and 4 μL of diluted cDNA template. PCR settings were 95 °C for 10 min, followed by 45 cycles of 95 °C for 10 s and 60 °C for 1 min, carried out on a Roche LightCycler 480 system. Melt curves were made using a gradual increase in temperature of 0.11 °C/s with five acquisitions per second and a temperature range of 60 to 90 °C. The melt curves were examined using the LightCycler 480 software (Roche). PCR efficiency was determined using LinRegPCR software (Supporting Information, TableS1). The resultant C_q values were exported and normalized to loading control (UniSp6; Exiqon), using the ΔΔC_t method. Cut‐off number of cycles was set as 40. The thresholds used to determine significance of differentially expressed miRNAs were set as P < 0.05 and a ΔΔC_t ≥ 1 cycle. The primers used are listed in Supporting Information, TableS2.

Candidate miRNAs were validated in the complete cohort of 45 patients (COPD, smoker, and non-smoker groups, n = 15 each). We used the miRCURY LNA Universal RT microRNA PCR System (Exiqon, Qiagen, Spain) for the detection of miRNAs by quantitative real-time PCR (qPCR) using SYBR green. According to the instructions of the reverse transcription reagent, 2 µL of total RNA was reverse transcribed into cDNA with miRCURY LNA RT Kit (Exiqon, Qiagen, Spain) and incubated for 60 min at 42 °C, 5 min at 95 °C to heat inactivate the reverse transcriptase, and stored at 4 °C. cDNA was diluted 20× with nuclease free water. Subsequent real-time PCR reactions were performed in duplicate with the miRCURY LNA SYBR Green PCR Kit (Exiqon, Qiagen, Spain) with LNA-specific primers using a CFX96 system (Bio-Rad, Sapin). The thermocycling conditions were as follows: 95 °C for 2 min, followed by 44 cycles of denaturation at 95 °C for 10 s, and annealing/extension at 56 °C for 1 min. Product specificity was confirmed in initial experiments by melting curve analysis, and PCR efficiency was calculated for each LNA specific primer. The expression of the housekeeping gene (miR-16-5p) was used as an internal control to normalize the expression of each target gene. Relative quantification with the 2^−∆∆Cq method [12 (link)] was used to evaluate the relative expression of miRNAs of interest.

Total RNA was extracted using the standard RNA extraction method with TRIzol ${}^{TM}$ (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). RNA concentration in each sample was assayed with a ND-1000 spectrophotometer (NanoDrop, Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription reactions were performed using the miRCURY LNA RT Kit (cod. 339340, Exiqon, Qiagen, Germany). Reverse transcription thermocycling parameters were as follows: 42 ${}^{\circ }$ C for 60 min and $95{}^{\circ }$ C for 5 min. Real time q-RT-PCR analysis was performed using ViiA 7 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). The PCR reaction included 1 ng of template cDNA, 1 $\mathsf{\mu }$ L of LNA PCR primers mix, 1 $\mathsf{\mu }$ L of RNAse-free water and 5 $\mathsf{\mu }$ L of miRCURY SYBR Green Master Mix (cod. 339345, Exiqon, Qiagen, Germany) in a total volume of 10 $\mathsf{\mu }$ L. Cycling conditions were as follows: 95 ${}^{\circ }$ C enzyme activation for 10 min, followed by 50 cycles of amplification: 10 ${}^{″}$ at 95 ${}^{\circ }$ C for denaturing, 1 min at 60 ${}^{\circ }$ C for annealing/elongation. Melting curve analysis was performed between 60 ${}^{\circ }$ C and 95 ${}^{\circ }$ C at a ramp rate of 0.11 ${}^{\circ }$ C/s.

For reverse transcription, 2 different platforms (1) for miRNAs (miRCURY LNA RT kit [Exiqon]) and (2) for lncRNAs and circRNAs (SuperScript VILO MasterMix [Invitrogen]) were used. For further details see Online Data Supplement.

Total exosomal miRNAs were reverse transcribed to cDNA using miRCURY LNA™ RT Kit (Exiqon) according to the manufacturer's instructions. Quantitative PCR was performed using miRCURY LNA™ SYBR Green Mix (Exiqon) in Vii7A real‐time PCR system (Applied Biosystems). The miRNA‐specific primer sequences were provided by Exiqon based on the miRNA sequences obtained from the miRBase database. At the end of the PCR cycles, melting curve analyses were performed. Fold changes in expression of each miRNA were calculated by a comparative threshold cycle (C_t) method using the formula:
2-[ΔCt(InternalControl)-ΔCt(sample)]. Currently, there is no standard internal control for exosomal miRNAs. MiRNAs with relatively abundant and consistent expressions released from profiling were applied as candidate internal controls.

Total RNA was extracted from rat PFC and primary neuronal cultures using Tri-Reagent (Sigma-Aldrich) and Direct-zol RNA MiniPrep (Zymo Research, Freiburg, Germany), in accordance with the instructions of the manufacturer [19 (link),32 (link)]. Reverse transcription was carried out using miRCURY^® LNA^® RT kit (Exiqon, QIAGEN, Milano, Italy). qPCR was performed using iTaq Universal SYBR Green supermix (Bio-Rad Laboratories, Milano, Italy). Primers used for qPCR are as follows: miR-135a-5p, YP00204762; SNORD68, YP00203911; RNU1A1, and YP00203909 (miRCURY^® LNA^® miRNA PCR assay, Exiqon). The relative expression of miRNAs was calculated using the comparative Ct (ΔΔCt) method and is expressed as fold change. The mean of SNORD68 and RNU1A1 was used as a control reference.