The expression level of miRNA-145 was assessed using SYBR Green qPCR analysis and normalized with SNORD68 in ovarian samples. RNA conversion to complementary DNA was carried out using miScript II RT Kit (Qiagen, Cat no. 218161) using thermocycler (Robocycler Gradient 96, BIOMETRA®, Princeton, NJ, USA). The following miScript primer assays (Qiagen, Cat no. 218300) were used for miRNA-145 (Thermofisher Scientific, Waltham, Ma, USA, MS00000434), and for SNORD68 (Thermofisher Scientific, USA, MS00033712). In brief, 12.5 μL of miScript SYBR Green PCR master mix (Qiagen, Cat no. 218073), 2.5 μL miScript primer Assays, 7.5 μL PCR grade water, and 2.5 μL cDNA template diluted to reach concentration of 20 ng cDNA were included in the reaction. The real-time PCR reactions were performed in accordance with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines [60 (link)]. Fold change of miRNA-145 was estimated using LIVAK method (=2−ΔΔCt).
Miscript primer
Manufactured by Qiagen
Sourced in United States, Germany, Netherlands
The MiScript primers are a set of oligonucleotide sequences designed for the detection and quantification of microRNA (miRNA) expression levels. These primers are specifically engineered to target and amplify miRNA sequences, which are essential regulatory molecules involved in various cellular processes. The MiScript primers provide a reliable and consistent tool for researchers to study miRNA expression patterns in their experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Miscript 2 rt kit, by Qiagen (8 mentions)
Miscript sybr green pcr kit, by Qiagen (5 mentions)
Mirneasy mini kit, by Qiagen (3 mentions)
Miscript reverse transcription kit, by Qiagen (2 mentions)
Cfx384 touch real time pcr machine, by Bio-Rad (2 mentions)
Mirna specific primer, by Qiagen (2 mentions)
Sybr green, by Roche (2 mentions)
Robocycler gradient 96, by Analytik Jena (1 mentions)
Miscript sybr green pcr master mix, by Qiagen (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Rna 6000 nano, by Agilent Technologies (1 mentions)
Miscript hiflex buffer, by Qiagen (1 mentions)
Quantitect primer assay, by Qiagen (1 mentions)
Mircute mirna extraction kit, by Tiangen Biotech (1 mentions)
Cfx96 real time detection system, by Bio-Rad (1 mentions)
Miscript universal primer, by Qiagen (1 mentions)
Quantitect sybr green pcr master mix, by Qiagen (1 mentions)
Rnase free dnase set, by Qiagen (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
20 protocols using miscript primer
1
Quantitative Analysis of miRNA-145 in Ovarian Samples
2
Extracting and Profiling miRNA via RT-qPCR
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Total RNA containing miRNAs was extracted by miRNeasy Mini kit (Qiagen), including a DNase step, according to the manufacturer’s instructions. The integrity of total RNA and miRNAs was checked by RNA 6000 Nano and Small RNA assays, respectively, on the 2100 Bioanalyzer (Agilent Technologies) and quantification performed by Nanodrop ND-1000 spectrophotometer (Thermo Scientific). Only RNAs with a RIN ≥7 were included in subsequent experiments. One μg of total RNA, containing miRNAs, was retro-transcribed using miScript Reverse Transcription Kit (Qiagen) and miScript HiFlex Buffer in a final volume of 20 μl. A poly(A) tag was added during the reaction and reverse transcription (RT) was performed in presence of both oligo-dT and random primers. The oligo-dT primers had a universal tag sequence on the 5′ end that, with a specific miScript primer (Qiagen) different for each miRNA, allowed amplification in the qPCR step.
3
Quantification of Serum and Hepatic miRNAs
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Serum and hepatic miRNAs were isolated and quantified using miRcute miRNA Extraction kit (TIANGEN, Beijing, China), first-strand cDNA synthesis, and quantitative PCR detection kits, in accordance with the manufacturer’s instructions. The expression level of mature miRNA was determined by qRT-PCR analysis using commercial miScript Primer or QuantiTect Primer Assays from Qiagen. Total HBV RNA was extracted and detected as described previously [18 (link)].
4
Quantification of shrimp miR-10a expression
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Total RNA isolated from shrimp stomach was treated by on-column DNase digestion (RNase-free DNase set; Qiagen) according to the manufacturer’s instructions. Reverse transcription was performed using the miScript II RT kit (Qiagen) according to the manufacturer’s instructions. Briefly, DNase-treated RNA was reverse transcribed into cDNA using the 5× miScript HiSpec buffer for quantification of mature miRNAs. miRNA expression was analyzed using the miScript SYBR Green PCR kit (Qiagen) on a CFX96 Real-Time Detection System (Bio-Rad) according to the manufacturer’s instructions. Each reaction consisted of 2.5 µl of diluted cDNA was mixed with 12.5 µl of 2× QuantiTect SYBR Green PCR Master Mix, 2.5 µl of 10× miScript Universal Primer, and 2.5 µl of 10× miScript Primer (specific for miR-10a or the U6 internal control, Qiagen).
5
Validating Altered miRNAs in Endometriosis and Ovarian Cancer
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For the validation of the most relevant altered miRNAs, we selected a subset of 17 normal samples, 33 endometriosis samples and 28 ovarian cancer samples. We used a two-step qRT-PCR. cDNA was generated using miScriptII RT Kit and then was diluted 1:5. The diluted cDNA sample was used as a template for performing qRT-PCR using QuantiTect (QuantiTect SYBR Green PCR kit, Qiagen) with specific miScript Primer (Qiagen). qRT-PCR assays were performed on ViiA 7 Real Time PCR System using the recommended amplification protocol (45 cycles of 30 seconds at 95°C, 30 seconds at 55°C and 30 seconds at 72°C) and a melting curve step.
6
Profiling miRNA and mRNA Expression in PBMCs
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We performed qPCR of specific miRNAs and mRNAs of interest. RNA was isolated from PBMCs and purified monocytes using the Direct-zol mini kit per the manufacturer's instructions (Zymo Research, Irvine, CA). For mRNA measurement, RNA was reverse transcribed using the Retroscript kit (Invitrogen, Carlsbad, CA) and measured with PowerSYBR qRT-PCR kits (Applied Biosystems, Foster City, CA) on aQuantStudio5 analyzer (Applied Biosystems, Foster City, CA). Relative messenger RNA (mRNA) expression was determined using gene-specific primers listed in Table 1 . Analyses were performed using the ddCT method (average Ct of gene of interest–average Ct of 18S). For miRNA measurements, RNA was reverse transcribed using the miScript II RT kit (Qiagen, Hilden, Germany) with the HiSpec buffer. Relative expression was measured using the miScript SYBR Green PCR kit with miScript primers (Qiagen, Hilden, Germany) for each miRNA analyzed. Statistical analyses were performed using the ddCT method (average CT of miRNA—average CT of control SNORD68).
7
miRNA Expression Analysis by RT-qPCR
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RT-qPCR was performed using the miScript PCR system (Qiagen, Hilden, Germany). For a subset of 87 samples (31 with future tumor and 56 controls) we determined the expression levels of three miRNAs, i.e., miR-150-5p, miR-574-3p, and miR-631. RNU6B and SNORD48 were used as endogenous controls. In detail, 100 ng total RNA was reversely transcribed using miScript RT II kit, and 1 ng cDNA was used for qPCR using miScript SYBR Green reagent and respective miScript Primers (Qiagen) on a StepOnePlus cycler (Applied Biosystems, Foster City, CA). All reactions were set up in duplicates. Data were analyzed using StepOneSoftware (Applied Biosystems).
8
Profiling miRNA and mRNA Expression
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Total RNA was isolated from tissue samples and cells using miRNeasy Mini Kit (Qiagen) and stored at −80°C. First-strand cDNA synthesis was performed with 0.5–1 µg total RNA using miScript II RT Kit (Qiagen) for miRNA analyses and TAKARA kit for mRNA expression analyses according to the manufacturer’s instructions (TAKARA). Real-time RT-PCR was performed on a Bio-Rad machine using Syber Green method (Primer sequences are shown in Table S2 in Supplementary Material). For miRNA detection, cDNA was amplified using miScript primers (Qiagen). miRNA expression data were normalized against snord68 and snord72 transcript levels (Qiagen) while β-actin and GAPDH genes were used to normalize mRNA expression.
9
Quantitative Analysis of mRNA and miRNA
10
Exosomal miRNA Expression Analysis
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miRNAs were isolated from exosomes (Exo-FBS and Exo-RA) and iMSCs using a Total Exosome RNA Protein Isolation Kit (Invitrogen, Waltham, Massachusetts, USA) and Tri-Reagent (MRC), respectively. cDNA was generated using the miRCURY LNA RT kit (Qiagen, Venlo, Netherlands), and qPCR was performed using the miRCURY LNA SYBR Green PCR kit with several miScript primers (Qiagen): hsa-miR-16a-5p, hsa-miR-155-5p, hsa-miR-146a-5p, hsa-miR-10a-5p, hsa-miR-142-3p, and hsa-miR-216a-5p. qPCR was performed according to the protocol provided by the manufacturer. miR-16-5p served as the housekeeping gene and the 2−ΔΔCT method was used to determine expression levels.
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