The miRNA expression analysis was performed in duplicate for the following miRNAs using miRCURY LNA miRNA PCR Assays (Qiagen): let-7b-5p, hsa-miR-140-3p, hsa-miR-9-5p, hsa-miR-16-5p, hsa-miR-21-5p, hsa-miR-25-5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-200a-3p, hsa-miR-203a-3p, hsa-miR-218-5p. U6 snRNA and SNORD48 were chosen for the normalization of miRNA expression. Real-time PCR was performed using the QuantStudio5 thermal cycler (Thermo Fisher Scientific). The reaction was performed using miRCURY LNA SYBR Green Master Mix (Qiagen) according to the manufacturer’s recommendations. The initial data analysis was prepared using QuantStudio Design & Analysis Software v1.5.1 (Thermo Fisher Scientific) to obtain raw Ct values. The relative quantification of miRNAs expression was determined.
Mircury lna sybr green master mix
Manufactured by Qiagen
Sourced in Germany, Denmark
The MiRCURY LNA SYBR Green Master Mix is a laboratory reagent designed for real-time quantitative PCR (RT-qPCR) analysis of microRNA (miRNA) expression. It contains a proprietary SYBR Green I-based detection system and Locked Nucleic Acid (LNA) technology to enable sensitive and specific quantification of miRNA targets.
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Lab products found in correlation
Mircury lna rt kit, by Qiagen (6 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Lightcyclerr 480 real time pcr system, by Roche (2 mentions)
Lc software 4, by Roche (2 mentions)
Lightcycler 480 real time pcr system, by Roche (2 mentions)
Mircury lna mirna pcr assay, by Qiagen (2 mentions)
Mircury lna universal cdna synthesis kit, by Qiagen (2 mentions)
Mir specific lna pcr primer set, by Qiagen (2 mentions)
Mircury lna universal rt microrna kit, by Qiagen (2 mentions)
Quantstudio 12k flex system, by Thermo Fisher Scientific (2 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Mircury lna universal rt microrna pcr assay, by Qiagen (2 mentions)
Quantstudio5 thermal cycler, by Thermo Fisher Scientific (1 mentions)
Quantstudio design analysis software v1, by Thermo Fisher Scientific (1 mentions)
Cfx96 or cfx384 real time pcr system, by Bio-Rad (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (1 mentions)
Tri reagent, by Molecular Research Center (1 mentions)
Mircury lna mirna serum plasma focus pcr panels, by Qiagen (1 mentions)
Mircury lna mirna pcr, by Qiagen (1 mentions)
17 protocols using mircury lna sybr green master mix
1
MicroRNA Expression Analysis Protocol
2
Quantitative miRNA Analysis in Plasma and Colonies
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miRCURY LNA RT Kit (QIAGEN, Hilden, Germany) was used to assay miRNA quantitative real–time polymerase chain reaction (qRT–PCR) in plasma and Hill colonies as described previously in detail [31 (link)]. Briefly, cDNA was diluted 100× and assayed in 10 μL PCR reactions according to the protocol for miRCURY LNA miRNA PCR; miRNA was assayed once by qPCR on the miRNA Ready-to-Use PCR, Human panel I + II (Catalog number: 339322, QIAGEN) using miRCURY LNA SYBR Green master mix. The amplification was performed in a LightCyclerR 480 Real-Time PCR System (Roche, Basel, Switzerland) in 384 well plates and analyzed using the Roche LC software 4 (Basel, Switzerland) both for the determination of Cq. All Cq data were normalized using the global mean method based on the average of the assays detected in all samples, yielding ΔCq values. Fold-change analysis was performed using 2 × |ΔΔCq| calculation, with ΔΔCq obtained from (∆Cq × T1DM) − (∆Cq × HCs).
3
Quantifying mRNA and miRNA Expression
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RNA was extracted from HLF-1 cells using the TRI Reagent® (Molecular Research Center, Inc.). cDNA synthesis was performed using a PrimeScript RT Reagent Kit (Takara Bio, Inc.) or miRCURY LNA RT kit (Qiagen GmbH). cDNA of mRNA was mixed with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories, Inc.) and primers (Table I ). cDNA of miRNA was mixed with miRCURY LNA SYBR Green Master Mix (Qiagen GmbH) and miR-6757-3p or U6 primers. Gene expression was assessed using CFX96™ or CFX384™ Real-Time PCR System and CFX Maestro version 3.1.1517.0823 software (Bio-Rad Laboratories, Inc.). The thermocycling protocol for mRNA consisted of an initial cycle at 95˚C for 3 min, followed by 50 cycles at 95˚C for 2 sec and 60˚C for 10 sec, and finally from 65˚C to 95˚C. The thermocycling protocol for miRNA consisted of an initial cycle at 95˚C for 10 min, followed by 50 cycles at 95˚C for 10 sec and 60˚C for 1 min, and finally 60˚C for 30 sec after increasing from 65˚C to 95˚C. mRNA expression was normalized to ACTB. miRNA expression was normalized to U6. Results are shown as relative values with the mean of the control or TGF-β + cont. mimic group set to 1 (except for miRNA data in Fig. 2B ).
4
Detailed miRNA Expression Analysis Protocol
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cDNA was prepared using the miRCURY LNA RT Kit (Qiagen) as per the manufacturer’s instructions. Total reaction volume was 20 μl, using 4 μl of RNA per reaction, and 1 μl of cel-miR-39-3p (0.002 fmol/μl) and 1 μl of UniSp6 included as positive controls. MicroRNA expression analysis was performed using the miRCURY LNA miRNA Serum/Plasma Focus PCR Panels (Qiagen). The panel consists of two 96-well plates and contains 179 pre-validated assays for miRNAs commonly present in human serum and plasma, as well as 7 potential reference genes in addition to assays for the spike in controls and cel-miR-39-3p. qPCR was performed using the ABI7500 FAST system as per the manufacturer’s instructions. Briefly, 20 μl of cDNA was combined with 2000 μl of miRCURY LNA SYBR Green Master Mix (Qiagen) and 10 μl was aliquoted per well across the two plates. The plates were sealed, spun briefly, and run consecutively on the same instrument.
5
Quantification of miRNA expression
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A total of 10 μL RNA was reverse transcribed in 50 μL reactions using the miRCURY LNA RT Kit (Qiagen, Hilden, Germany). Complementary deoxyribonucleic acid (cDNA) was diluted 50× and assayed in 10 μL polymerase chain reactions (PCR) according to the protocol for miRCURY LNA miRNA PCR (Qiagen, Hilden, Germany). The expression level of 372 miRNAs was assayed once by quantitative PCR (qPCR) on the miRNA Ready-to-Use PCR, Human panel I (Qiagen, Hilden, Germany; see Supplementary Table S1 for the list of assays) using miRCURY LNA SYBR Green master mix (Qiagen, Hilden, Germany). Negative controls excluding template from the reverse transcription reaction was performed and profiled similar to the samples. The amplification was performed in a LightCycler® 480 Real-Time PCR System (Roche, Basel, Switzerland) in 384 well plates. Fluorescence data were converted into quantification cycle (Cq).
6
Reverse Transcription and qPCR for miRNA Analysis
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10ng of total RNA were reverse-transcribed and amplified using the miRCURY LNA miRNA PCR Starter Kit (Qiagen, Ref 339320. The kit includes a spike-in control primer set (UniSp6), UniSP6 RNA Spike-in-template, one candidate endogenous control primer set (miR-103a-3p) and two validated primer sets, which in our case were miR-297 (Qiagen YP00206079) and miR-574-5p (Qiagen YP02116206). As additional control micro-RNAs (miRNAs), we chose miR-25-3p (Qiagen YP00204361) and miR-331-3p (Qiagen YP00206046) because both are used as markers in BC analysis (27 (link), 28 (link)) and their expression is not modified by RA treatment in the BC cell line SKBR3 (11 (link)). Real-time PCR was performed with miRCURY LNA SYBR Green Master Mix (Qiagen) in 10μL total volume using the CFX 96 Real Time System (Bio-Rad). The expression of target miRNAs miR-297 and miR-574-5p was normalized against miR-25-3p, miR-103a-3p, miR-331-3p and UniSp6 using the 2-ΔΔCt method. To validate the real-time system used for miRNA analysis, we measured the levels of UniSp6RNA, a control RNA provided with the Starter Kit, that was added before the reverse transcription in equal amount to all samples (see Supplementary material).
7
Exosomal miRNA Profiling by RT-qPCR
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Reverse transcription was performed using the miRCURY LNA Universal RT miRNA Kit (Qiagen, Germantown, MD) according to the manufacturer’s instructions. Briefly, 4uL of exosomal RNA was polyadenylated and converted to cDNA. A total of 0.5uL of UniSp6 RNA was spiked in the RT mix for cDNA synthesis quality control. Total reaction volume was 10uL. Reaction temperature cycling consisted of an RT step at 42°C for 60 minutes, inactivation at 95°C for 5 minutes, and immediate cooling to 4°C. Real-time qPCR was performed using miRCURY LNA SYBR® Green master mix and miRCURY LNA miRNA PCR Assay (Qiagen, Germantown, MD). The miRNA specific primers sets were as follows: hsa-miR-122-5p: 5’-UGGAGUGUGACAAUGGUGUUUG-3’ , hsa-miR-16-5p: 5’ UAGCAGCACGUAAAUAUUGGCG-3’ . All PCR assays were carried out on a QuantStudio 5 Flex Real-Time PCR System (Applied Biosystems. Inc, Foster City, CA) programmed as follows: 95°C for 2 min, followed by 40 cycles of 95°C for 10 s and 56°C for 1 min. Real-time qPCR assays (total reaction volume 10uL) for each target miRNA were set up in triplicate reactions. No-template controls were included. The comparative cycle threshold (CT) was used to evaluate the relative detection level of miR-122 in each sample and expression levels were normalized to miR-16-5p. Fold-change was calculated using the delta-delta CT method (2−ΔΔCT) of relative quantification.
8
Profiling miRNA Expression in Regeneration
9
Profiling Serum miRNA in Endometriosis
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Serum samples from women with endometriosis (n = 10) and from the control group (n = 10) were analyzed using the miRNA Ready-to-Use PCR. A total of 185 miRNAs were evaluated by qPCR in serum using the miRCURY LNA SYBR Green master mix (QIAGEN, Hilden, Germany). Amplification was performed with a LightCycler® 480 real-time PCR system (Roche, Mannheim, Germany) in 384-well plates. Amplification curves were analyzed using Roche LC 480 software for both Cq value determination and melting curve analysis. Normalization was performed based on the average of the assays detected in all samples (105 miRNAs), according to the following formula: normalized Cq (dCq) = global mean Cq − assay Cq (miRNA of interest). A higher value thus indicated that the miRNA was more abundant in the sample. The 2nd derivate method was used to analyze the relative changes in gene expression from expression profiling, obtaining the fold change.
10
Quantifying miRNA Differential Expression
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Total RNA was isolated using the miRNeasy Micro Kit (QIAGEN, Hilden, Germany), as explained in Phowira et al., 2022 [8 (link)].
Reverse transcription was performed using the miRCURY LNA RT Kit (QIAGEN, Hilden, Germany) on about 10 μL of RNA. Following the methodology for miRCURY LNA miRNA PCR, cDNA was diluted 100× and assessed in 10 μL PCR reactions. The miRNA was assayed once by qPCR using the miRCURY LNA SYBR Green master mix on the miRNA Ready-to-Use PCR, Human panel I + II (Catalog number: 339322, QIAGEN). The amplification was carried out using a LightCyclerR 480 Real-Time PCR System (Roche, Basel, Switzerland) and data were analyzed using Roche LC software 4 (Basel, Switzerland). The ΔCq values were obtained by using the global mean normalization approach to correct all Cq data. Fold-change analysis was performed using 2 × |ΔΔCq| calculation, with ΔΔCq obtained from (∆Cq × T1DM) − (∆Cq × HCs).
Reverse transcription was performed using the miRCURY LNA RT Kit (QIAGEN, Hilden, Germany) on about 10 μL of RNA. Following the methodology for miRCURY LNA miRNA PCR, cDNA was diluted 100× and assessed in 10 μL PCR reactions. The miRNA was assayed once by qPCR using the miRCURY LNA SYBR Green master mix on the miRNA Ready-to-Use PCR, Human panel I + II (Catalog number: 339322, QIAGEN). The amplification was carried out using a LightCyclerR 480 Real-Time PCR System (Roche, Basel, Switzerland) and data were analyzed using Roche LC software 4 (Basel, Switzerland). The ΔCq values were obtained by using the global mean normalization approach to correct all Cq data. Fold-change analysis was performed using 2 × |ΔΔCq| calculation, with ΔΔCq obtained from (∆Cq × T1DM) − (∆Cq × HCs).
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