For miRNAs, total miRNAs were isolated by miRNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. First-strand cDNA was synthesized using random oligonucleotides and a miRcuRY LNA Universal RT microRNA PCR Universal cDNA Synthesis Kit II (EXIQON, USA) and U6 was employed as an internal reference. Primers (Table S5) were designed by using miRprimer and the optimum primer pairs were selected [36 (link)]. RT-qPCR was performed by using miRcuRY LNA Universal RT microRNA PCR Exilent SYBR master mix (EXIQON) and a StepOne Plus system. For mRNAs, total RNAs were isolated and cDNAs were synthesized. RT-qPCR was performed by using GoTaq®qPCR Master Mix (Promega) and the Roche LightCycler® 480 system.
Mircury lna universal rt microrna pcr universal cdna synthesis kit 2
Manufactured by Qiagen
Sourced in United States
The MiRCURY LNA™ Universal RT microRNA PCR/Universal cDNA Synthesis Kit II is a laboratory equipment product designed for the reverse transcription and real-time PCR quantification of microRNA. The kit enables the synthesis of complementary DNA (cDNA) from microRNA samples and the subsequent real-time PCR amplification and detection of the cDNA.
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Lab products found in correlation
Mirneasy mini kit, by Qiagen (3 mentions)
Mircury lna universal rt microrna pcr exilent sybr green master mix, by Qiagen (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Miscript sybr green pcr kit, by Qiagen (2 mentions)
Gotaq qpcr master mix, by Promega (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
Qx200 droplet digital pcr system, by Bio-Rad (1 mentions)
Warmstart rtx reverse transcriptase, by New England Biolabs (1 mentions)
Nucleospin mirna, by Takara Bio (1 mentions)
Primescript reverse transcriptase, by Takara Bio (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Abi 7000 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Exilent sybr green master mix, by Qiagen (1 mentions)
Tripure isolation reagent, by Roche (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
Lightcycler 480 sybr green 1 master mix, by Roche (1 mentions)
High capacity cdna archive kit, by Thermo Fisher Scientific (1 mentions)
9 protocols using mircury lna universal rt microrna pcr universal cdna synthesis kit 2
1
Quantifying miRNA and mRNA Levels
2
Digital Droplet PCR for EV Quantification
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Reverse transcription was performed using miRCURY LNA Universal RT microRNA PCR, Universal cDNA Synthesis Kit II (Exiqon) following the manufacturer's instructions with the following reaction composition: 2.3 μl of 5× reaction buffer, 1.15 μl enzyme mix, 0.5 μl synthetic RNA spike-in and 7.5 μl of template total RNA.
QX200TM Droplet DigitalTM PCR System (BioRad) was used to quantify mRNA copies using EvaGreen chemistry and the following primers: 5′-CAACGAATTTGGCTACAGCA-3′ and 5′-AGGGGTCTACATGGCAACTG-3′ for GAPDH; 5′-GATTTTGGTCTAGCTACAGA and 5′-GGATTTTTATCTTGCATTC for BRAF.
The direct encapsulation of EVs into oil droplets has been performed using isolated EVs as input sample, EvaGreen master mix supplemented with 5 U/ml of WarmStart® RTx Reverse Transcriptase (NEB) and with 3 mM magnesium sulphate. The standard protocol of ddPCR included a starting 10 min step at 55 °C followed by 5 min at 65 °C.
We optimized the procedure working with 102–106 polydisperse EVs to obtain at least 15,000 droplets in each assay.
QX200TM Droplet DigitalTM PCR System (BioRad) was used to quantify mRNA copies using EvaGreen chemistry and the following primers: 5′-CAACGAATTTGGCTACAGCA-3′ and 5′-AGGGGTCTACATGGCAACTG-3′ for GAPDH; 5′-GATTTTGGTCTAGCTACAGA and 5′-GGATTTTTATCTTGCATTC for BRAF.
The direct encapsulation of EVs into oil droplets has been performed using isolated EVs as input sample, EvaGreen master mix supplemented with 5 U/ml of WarmStart® RTx Reverse Transcriptase (NEB) and with 3 mM magnesium sulphate. The standard protocol of ddPCR included a starting 10 min step at 55 °C followed by 5 min at 65 °C.
We optimized the procedure working with 102–106 polydisperse EVs to obtain at least 15,000 droplets in each assay.
3
Quantitative RNA Expression Analysis in HeLa Cells
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RNA was extracted from HeLa cells using the RNeasy Mini kit (Qiagen, Hilden, Germany), and cDNA was generated using PrimeScript reverse transcriptase (Takara). Small RNA was extracted from RIP assay using NucleoSpin miRNA (Takara), and cDNA was generated using miRCURY LNA™ Universal RT microRNA PCR Universal cDNA synthesis KitII (EXIQON, Copenhagen, Denmark). PCR was performed using the SYBR® Premix Ex Taq™ II (Takara) (for mRNA) or miRCURY LNA™ Universal RT microRNA PCR ExiLENT SYBR® Green master mix (EXIQON) (for small RNA). The relative RNA expression levels were normalized to GAPDH or miRCURY LNA UniRT PCR Control primer mix UniSp6 (203954, EXIQON). The sequences of primers used to amplify each gene were: 5′-AGGTGGAGGAGTGGGTGTCGCTGTT-3′ and 5′-CCGGGAAACTGTGGCGTGATGG-3′ (GAPDH); 5′-CAGCATCCACAGTGCAGATC-3′and 5′-GCACCTCAGAGAAGCAATGC-3′(NOP56); 5′-TGGCAGCTATGTGTCTTGGA-3′and 5′-TGCCAGCCATACCATTCTCT-3′(NOP58); 5′-TGCTCTGATGAAATCACTAA-3′and 5′-AATCAGACAGGAGTAGTCTT-3′(SNORD49A); miRCURY LNA UniRT PCR Reference primer mix SNORD44 (203902, EXIQON).
4
Quantifying miRNA Expression by RT-qPCR
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Total RNA from tissues or cell lines was extracted using miRNeasy Mini kits (Qiagen, Crawley, UK). Target miRNAs were reverse transcribed to cDNA using gene-specific RT primers with the miRCURY LNA™ Universal RT microRNA PCR/Universal cDNA Synthesis Kit II (Exiqon, Woburn, MA, USA). miRNA expression profiles of tissues or cell lines were determined using the miScript SYBR Green PCR Kit (Qiagen) with the ABI7000 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). Primers for miRNA quantification were miRCURY LNA Universal primer (hsa-miR-410, hsa-miR-214, and hsa-miR-495) and U6 snRNA PCR primer (Exiqon). The relative quantification value of the target, normalized to the U6 snRNA control, was calculated by the comparative Ct method.
5
Quantitative Gene Expression Analysis
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Total RNA was extracted from pancreas, liver, and heart using Tripure Isolation Reagent (Roche) and Rneasy Mini Kit (Qiagen) and from isolated islets with Rneasy Micro Kit (Qiagen). Total RNA was reverse-transcribed with Transcriptor First Strand cDNA Synthesis Kit (Roche). qPCR was performed in a LightCycler® 480 II (Roche) using LightCycler® 480 SYBR Green I Master mix (Roche). Primer sequences are listed in Supplemental Table 2 . Results were analyzed using the Pfaffl's mathematical model [30] (link) and values were normalized to murine Rplp0 expression. miRNA-enriched RNA was extracted using miRVana™ miRNA Isolation Kit (Life Technologies). Ten nanograms of RNA were reverse-transcribed with miRCURY LNA™ Universal RT microRNA PCR – Universal cDNA synthesis kit II (Exiqon). Expression was quantified by qPCR using ExiLENT SYBR® Green Master mix (Exiqon) relative to U6snRNA expression.
6
Reverse Transcription and qPCR Protocol for microRNA
7
Real-time qPCR Gene Expression Profiling
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The reactions were performed in triplicate on an ABI PRISM 7900 real-time sequence detection system (Life Technologies). The results are presented as relative quantification (RQ) and are calculated as RQ=(2−ΔΔCt) ± RQ min/max when using the control samples as calibrators or as 2−ΔCt ± SD for direct comparisons. miRVANA (Ambion) and a High-Capacity cDNA Archive kit (Life Technologies) or Mircury LNA Universal RT microRNA PCR universal cDNA synthesis kit II (Exiqon) were used for RNA extraction and retrotranscription. TaqMan assays for CCL2 (Hs00234140_m1), HIF1A (Hs00153153_m1) and β-actin (4326315E) were used, and Exiqon reagents were used for miRNA amplifications.
8
Serum miRNA Isolation and Quantification
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Serum samples were obtained from TSC patients using standard vials with clotting activator (S-Monovette ® 2.7 ml). After clot formation, samples were centrifuged at 2500 rpm for 10 min. Afterwards, serum was collected and stored at -80°C until testing. Total RNA including the miRNA fraction was isolated from 200 μl of serum samples using miRCURY RNA Isolation Kit -Biofluid (Exiqon) according to the manufacturer's instructions. Purified RNA was eluted in 50 μl of H 2 O. Equal volume of each RNA sample (4 μl) was reverse transcribed in 20 μl RT reaction using miRCURY LNA Universal RT microRNA PCR -Universal cDNA Synthesis Kit II (Exiqon, 203301) according to the manufacturer's instructions.
9
miRNA-211 Expression Analysis in Daoy Cells
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Total RNA from Daoy cells was extracted using miRNeasy Mini kits (Qiagen, Crawley, UK). miR-211 was reverse transcribed to cDNA using gene-specific RT primers with the miRCURY LNA™ Universal RT microRNA PCR/Universal cDNA Synthesis Kit II (Exiqon, Woburn, MA, USA). miR-211 expression was determined using the miScript SYBR Green PCR Kit (Qiagen) with the Stratagene Mx3000P real-time PCR system (Stratagene, La Jolla, California). The relative quantification of miR-211 was normalized to the U6 snRNA and was calculated by 2 -∆∆Ct method [19] .
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