Mircury lna sybr green pcr kit

Total RNAs were extracted from 4 ~ 6 samples for each of tears and sera (300 μL) using QIAzol (QIAGEN, Hilden, Germany) and miRNeasy Serum/Plasma Kit (Qiagen). To normalize for technical variation, 0.01 fmol of synthetic cel-miR-39-3p (QIAGEN) was spiked into each tear and serum sample after addition of denaturing solution. Total RNAs were treated with SUPERase-In (Thermo Fisher Scientific, Waltham, MA), and the quality and quantity of the extracted miRNAs were assessed using a spectrophotometer (NanoVue™; GE Healthcare, Little Chalfont, UK).
Four miRNAs, expressed at high levels in microarrays, were measured by quantitative polymerase chain reaction (qPCR) following the manufacturer’s instructions. Briefly, 1.12 μL of template RNAs were reverse-transcribed using miRCURY LNA RT Kit (QIAGEN), then real-time PCR was performed using miRCURY LNA SYBR Green PCR Kit (QIAGEN). The following primers were used: hsa_miR-184 miRCURY LNA miRNA PCR Assay, hsa_miR-3616-3p miRCURY LNA miRNA PCR Assay, hsa_miR-3610 miRCURY LNA miRNA PCR Assay, hsa_miR-203a-3p miRCURY LNA miRNA PCR Assay (QIAGEN). Fluorescence was monitored on a 7500 Real Time PCR system (Thermo Fisher Scientific). Relative expression levels were normalized to cel-miR-39-3p and calculated using standard curve method.

Total RNA was extracted from immune cells both from HFD and ND and 3T3-L1 differentiated adipocytes with the miR-10a-3p mimic and control miR (scramble) using the QIAGEN RNeasy mini kit (Cat. no. 74104). 1μg of extracted RNA from each sample was used for reverse-transcription into cDNA with an iScript cDNA synthesis kit (Cat. no. 1708891; Bio-Rad) or for miRs, a miRCURY LNA rt Kit (339340, QIAGEN, USA) according to the manufacturer’s protocols. Reverse-transcription quantitative polymerase chain reaction analysis (RT-qPCR) was performed using the iTaq Universal SYBR Green Supermix (Cat. 1725121; Bio-Rad, USA) or for miRs, the miRCURY LNA SYBR Green PCR Kit (339346, QIAGEN). Primers were purchased from Integrated DNA Technologies (IDT; Coralville, IA) and QIAGEN. The primer sequences for genes and catalog no of miR primers (from QIAGEN) are shown in Tables 1 and 2, respectively.

The quantification of miRNAs in bovine milk sEVs was performed using the miRCURY LNA RT Kit (339340, Qiagen) and miRCURY LNA SYBR Green PCR Kit (339346, Qiagen). Briefly, the RNA concentration of all milk sEVs was adjusted to 20 ng/µL, subjected to reverse transcription in a 10 µL reaction solution, and incubated for 60 min at 42 °C, followed by further incubation at 95 °C for 5 min to inactivate the reverse transcriptase enzyme. Primers such as bta-miR-29a, bta-miR-200a, bta-miR-26b, bta-miR-27b, and bta-miR-30b-5p were contained in the miRCURY LNA miRNA PCR Assay components (339306, Qiagen). For the qPCR, 3 µL complementary DNA (cDNA) was added to the total volume of the reaction mixture. The qPCR was performed using a StepOnePlus analytical thermal cycler (Applied Biosystems, Foster City, CA, USA). The thermal cycling program was as follows: 95 °C for 2 min for initial heat activation, followed by 40 cycles of 95 °C for 10 s for denaturation, and 56 °C for 1 min for annealing and extension. The melting curve was analyzed at 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. For each candidate internal control miRNA, the qPCR was performed in duplicates (technical replicates). The qPCR excel data for each candidate internal control miRNA were extracted and analyzed further.

For verification of the miRNA PCR array results and further functional experiments on miR-425-5p, isolated RNA samples with an enriched fraction of miRNAs were reverse transcribed utilizing the miRCURY LNA RT Kit (339340, Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Quantitative real-time PCR was performed utilizing the miRCURY LNA SYBR^® Green PCR Kit (339345, Qiagen, Hilden, Germany), according to the manufacturer’s instructions. As miRNA primers, hsa-miR-17-5p miRCURY LNA miRNA PCR Assay (YP02119304, 339306, Qiagen, Hilden, Germany), hsa-miR-425-5p miRCURY LNA miRNA PCR Assay (YP00204337, 339306, Qiagen, Hilden, Germany), hsa-miR-223-3p 5p miRCURY LNA miRNA PCR Assay (YP00205986, 339306, Qiagen, Hilden, Germany), and hsa-miR-7a-5p miRCURY LNA miRNA PCR Assay (YP00205727, 339306, Qiagen, Hilden, Germany) were used. For normalization, miR-24-5p (YP00203954, 339306, Qiagen, Hilden, Germany) and UniSp6 (YP00203954, 339306, Qiagen, Hilden, Germany) were used. Relative gene expression was calculated utilizing the 2^−∆∆Ct method.

Total RNA was extracted with Tri-Reagent (Thermo Fisher Scientific, AM9738). The RNA was reverse transcribed using miRCURY LNA RT Kit (Qiagen, 339340) for cDNA synthesis reactions, according to manufacturer’s protocol. Quantitative RT-PCR analysis of miRNAs was performed using miRCURY LNA SYBR Green PCR Kit (Qiagen, 339346), according to manufacturer’s protocol. Gene expression levels were quantified using primers for: hsa-miR-127-3p (Qiagen, YP00204048), hsa-miR-409-3p (Qiagen, YP00204358), hsa-miR-411-5p (Qiagen, YP00204531), hsa-miR-493-3p (Qiagen, YP00204557). Normalization was done with U6 snRNA (Qiagen, YP00203907). The 2^–ΔΔCt was used determined using ABI 7500 instrument (Applied Biosystems) to calculate the relative expression of each gene.

The miRNA validation cohort consisted of 74 controls (31 samples already used for NGS and 43 newly collected samples), 26 BD patients (18 samples already used for NGS and 8 newly collected samples), and 84 MDD patients (44 samples already used for NGS and 40 newly collected samples). The quantitative real-time PCR analysis was carried out on 7900HT Fast Real-Time PCR System (Applied Biosystems, USA) using miRCURY LNA™ SYBR Green PCR Kit (Qiagen, USA) according to the manufacturer's protocol. The following Qiagen's miRCURY miRNA Assays were used: hsa-let-7f-5p (YP00204359), hsa-let-7e-5p (YP00205711), hsa-miR-103a-3p (YP00204063), hsa-miR-125a-5p (YP00204339), hsa-miR-139-3p (YP00205661), hsa-miR-425-5p (YP00204337), hsa-miR-483-5p (YP00205693), and UniSp-100 assay from QIAseq miRNA Library QC qPCR Assay kit. Expression levels of target miRNAs were normalized using 2^-∆∆CT method [52 (link)] with hsa-miR-425-5p, hsa-miR-103a-3p, and UniSp-100 spike-in as recommended in the manufacturer's manual.

Total RNA was extracted from frozen placenta villous tissue using miRNeasy Mini Kit (Qiagen, Cat No- 217004) following the manufacturer’s instruction as described above. 10 ng of RNA was reverse-transcribed into cDNA at 42°C for 1 h for each sample using the miRCURY LNA RT Kit (Qiagen, Cat No- 339340) following the manufacturer’s instruction. For qPCR, the template cDNA from each sample was diluted at 1 in 60 in nuclease-free water and 6 µl of this diluted cDNA was used for qPCR reaction using miRCURY LNA SYBR Green PCR kit (Qiagen, Cat No- 339346) and miRCURY LNA miRNA PCR Assay primers (Supplementary Table S2) following the manufacturer’s instruction. Each qPCR reaction volume was 20 µl and the thermocycler (Rotor Gene Q) protocol consists of an initial heat inactivation at 95°C for 2 min followed by 40 cycles of denaturation at 95°C for 10 s and a combined annealing/extension at 56°C for 1 min, and a final melting curve analysis (64°C–95°C). U6 snRNA primer was used as internal control and normalisation which was expressed equally across the samples (Supplementary Figure S3). The Cq values were used to calculate the relative miRNA expression (2^−ΔCq, relative to U6 snRNA) and were presented as Log₁₀ expression in each group.