Lna primers

Manufactured by Qiagen

Sourced in Denmark

LNA primers are a class of nucleic acid analogs designed to improve the specificity and sensitivity of DNA/RNA detection and quantification. They contain locked nucleic acid (LNA) modifications that increase binding affinity and thermal stability, enabling more precise and reliable target identification.

Automatically generated - may contain errors

Lab products found in correlation

Universal cdna synthesis kit 2, by Qiagen (4 mentions) Universal cdna synthesis kit, by Qiagen (3 mentions) Steponeplus real time pcr system, by Thermo Fisher Scientific (3 mentions) Mircury lnatm universal rt microrna pcr kit, by Qiagen (2 mentions) Exilent sybr green master mix kit, by Qiagen (2 mentions) Mircury rna isolation kit, by Qiagen (2 mentions) Geneglobe, by Qiagen (2 mentions) Mircury lna universal rt mirna pcr system, by Qiagen (2 mentions) Mircury isolation kit, by Qiagen (2 mentions) Iq sybr green supermix kit, by Bio-Rad (2 mentions) Nanodrop, by Thermo Fisher Scientific (2 mentions) Qbase software, by CellCarta (2 mentions) Iscript cdna synthesis kit, by Bio-Rad (1 mentions) Genex software, by Qiagen (1 mentions) Turbo dnase, by Thermo Fisher Scientific (1 mentions) Sybr green master mix, by Thermo Fisher Scientific (1 mentions) 7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions) Genematrix universal rna purification kit, by EURx (1 mentions) Ng dart rt kit, by EURx (1 mentions) Phire hot start 2 dna polymerase, by Thermo Fisher Scientific (1 mentions)

18 protocols using lna primers

Validation of RNA Expression Changes

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Validation of changes in RNA expression (19 (link)) of selected markers was performed by reverse transcription followed by quantitative PCR (RT-qPCR) from rats used for miRNA-seq (first batch) as well as from independent experimental batches. miRCURY LNATM Universal RT microRNA PCR kit (for miRNA, Exiqon, Vedbaek, Denmark) and iScript^™ cDNA synthesis kit (for mRNA, Bio-Rad^® Laboratories Inc., Hercules, CA) were used for reverse transcription. LNA-primers (for miRNA, Exiqon), IDT PrimeTime qPCR primers [for mRNA, Integrated DNA Technologies (IDT^®), Coralville, IA] and SYBR^® Green Mastermix (BioTools Inc., Jupiter, FL) were used for qPCR. The relative expression of target RNA to references (miRNA to miR-16–5p and mRNA to b-actin) were calculated by the formula: 2^−ΔCT (ΔCT=CT (target-reference)). Data were then normalized to the mean relative expression of controls, to determine the fold change after irradiation.

Klein C.A., Schmidt-Kittler O., Schardt J.A., Pantel K., Speicher M.R, & Riethmüller G. (1999). Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells. Proceedings of the National Academy of Sciences of the United States of America, 96(8), 4494-4499.

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Quantitative Real-Time PCR of Specific miRNAs

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Real-time PCR was performed using the ExiLENT SYBR® Green Master Mix kit (Exiqon) for miR-96, miR-484, and miR-425 according to the manufacturer's instructions. Locked nucleic acid (LNA) primers (Exiqon) were used in all the experiments. cDNA was diluted 10x and added to the PCR reactions. A two-step real-time PCR protocol was performed using an initial denaturation step at 95°C for 10 min, 45 amplification cycles including a denaturation step (10 s at 95°C), and an annealing step (60 s at 60°C). A melting curve was determined for each reaction to confirm the precision of the reactions. Expression levels of all the miRNAs were normalized to the level of U6 as a control using the 2^–ΔΔCt method.

Alipoor S.D., Tabarsi P., Varahram M., Movassaghi M., Dizaji M.K., Folkerts G., Garssen J., Adcock I.M, & Mortaz E. (2019). Serum Exosomal miRNAs Are Associated with Active Pulmonary Tuberculosis. Disease Markers, 2019, 1907426.

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Profiling mRNA and miRNA Transcripts

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For mRNA analysis, total cellular RNA was isolated using the GeneMATRIX Universal RNA Purification Kit (Eurx, Gdansk, Poland) including a DNA digestion step with the Turbo DNAse (Ambion/Thermo Fisher Scientific) and reverse transcribed with the NG dART RT Kit (Eurx) according to the manufacturer’s recommendation.
For miRNA analysis, cellular RNA was isolated with the Total RNA isolation Kit (Exiqon, Vedbaek, Denmark) with the use of Turbo DNAse (Ambion) and transcribed to complementary DNA (cDNA) with the Universal cDNA Synthesis Kit II (Exiqon) following the vendor’s recommendation.
Transcript levels were measured using the real-time PCR method with the SYBR Green Master Mix (Applied Biosystems/Thermo Fisher Scientific) and specific primer sets (Supplementary Table S1). miRNA expression was analyzed with custom-designed plates containing the miRNA locked nucleic acid (LNA)™ primers (Exiqon). Quantification of mRNA/miRNA content was performed on the 7500Fast Real-Time PCR System (Applied Biosystems) using the ΔΔCt method. Gene expression levels were calibrated with a housekeeping gene—β-2-microglobulin. miRNA content in EVs was standarized with has-miR-103-3p, identified as a normalizer by the Norm Finder tool from the GenEx software (Exiqon).

Bobis-Wozowicz S., Kmiotek K., Kania K., Karnas E., Labedz-Maslowska A., Sekula M., Kedracka-Krok S., Kolcz J., Boruczkowski D., Madeja Z, & Zuba-Surma E.K. (2016). Diverse impact of xeno-free conditions on biological and regenerative properties of hUC-MSCs and their extracellular vesicles. Journal of Molecular Medicine (Berlin, Germany), 95(2), 205-220.

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Determination of HTT Haplogroups

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HTT haplogroups A, B, and C were defined using 2 intragenic SNPs, rs762855 and rs4690073, and haplotype A variants were further defined using 4 additional intragenic SNPs, rs2857936, rs363096, rs2276881, and rs362307.^{10 (link)} The SNPs were determined with restriction fragment analysis (FastDigest; Thermo Fisher Scientific, Waltham, MA), allele-specific amplification using locked nucleic acid (LNA) primers (Exiqon, Vedbaek, Denmark), or by sequencing (ABI3500xL Genetic Analyzer, Applied Biosystems, Foster City, CA) as appropriate (table e-1, links.lww.com/NXG/A153). The PCRs were performed using Phire Hot Start II DNA Polymerase (Thermo Fisher Scientific) according to the standard procedures. The LNA amplifications were conducted in duplicates or confirmed by sequencing. Additional SNPs (table e-1, links.lww.com/NXG/A153) were analyzed in samples, where haplogroup A variant remained undetermined.^{10 (link),14 (link)} Haplotypes were manually annotated and then phased to CAG repeat length by familial relationship or by homozygosity of haplotype.

Ylönen S., Sipilä J.O., Hietala M, & Majamaa K. (2019). HTT haplogroups in Finnish patients with Huntington disease. Neurology: Genetics, 5(3), e334.

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miRNA Expression Analysis Protocol

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First strand synthesis was executed using Universal cDNA synthesis kit (Exiqon) according to the manufacturer's instructions. Owing to its superior sensitivity, the SYBR Green master mix and specific-miRNA (miR-17 and miR-20a) LNA primers (Exiqon) were used according to the manufacturer's instructions for microarray validation and detection of specific miRNAs. Detection was carried out using the LC480 qPCR machine (Roche) according to the manufacturer's guidelines, followed by melting curve analysis at the end of the run.

Greenberg E., Hajdu S., Nemlich Y., Cohen R., Itzhaki O., Jacob-Hirsch J., Besser M.J., Schachter J, & Markel G. (2014). Differential regulation of aggressive features in melanoma cells by members of the miR-17-92 complex. Open Biology, 4(6), 140030.

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Real-time PCR for miRNA Expression Analysis

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Real-time PCR assays were performed using the ExiLENT SYBR® Green Master Mix Kit (Exiqon). LNA primers were purchased from Exiqon. cDNA was diluted 10x and added to the PCR reactions according to the manufacturer's instructions. The real-time PCR program included the following steps: an initial denaturation step at 95°C for 10 min and 50 cycles of amplification that consisted of a denaturation step (10 s at 95°C) and an annealing step (60 s at 60°C). The expression levels of miR-208b were normalized to the level of miR-16 as control using the efficiency-corrected calculation models of the Pfaffle method [29 (link)].

\begin{matrix} R a t i o = \frac{(E t a r g e t) Δ C t t a r g e t (c o n t r o l - s a m p l e)}{(E R e f) Δ C t R e f (c o n t r o l - s a m p l e)} . \end{matrix}

Alavi-Moghaddam M., Chehrazi M., Alipoor S.D., Mohammadi M., Baratloo A., Mahjoub M.P., Movasaghi M., Garssen J., Adcock I.M, & Mortaz E. (2018). A Preliminary Study of microRNA-208b after Acute Myocardial Infarction: Impact on 6-Month Survival. Disease Markers, 2018, 2410451.

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miRNA Extraction and Quantification

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Total RNA was extracted using a miRCURY RNA Isolation Kit (Exiqon, Vedbaek, Denmark)
and reverse transcribed using a Universal cDNA Synthesis kit or Universal cDNA
Synthesis kit II (Exiqon) according to manufacturer protocols. Real-time PCR analyses
were performed using Power SYBR Green PCR master mix (Applied Biosystems) and ABI
Step One real-time PCR systems (Applied Biosystems) according to the manufacturer
protocols. Expression levels of miRNA were normalized to the levels of U6 snRNA
(Rnu6) using the 2^–ΔΔCt method [38 (link)]. The primers used for real-time RT PCR analysis for miRNA
detection (LNA primers) were purchased from Exiqon.

SUMITOMO J.I., EMORI C., MATSUNO Y., UENO M., KAWASAKI K., ENDO T.A., SHIROGUCHI K., FUJII W., NAITO K, & SUGIURA K. (2016). Mouse oocytes suppress miR-322-5p expression in ovarian granulosa cells. The Journal of Reproduction and Development, 62(4), 393-399.

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Quantitative RNA Isolation and Analysis

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Total RNA was extracted from cells/tissue using the miRCURY isolation kit (Exiqon, Denmark), according to the manufacturer’s recommendations. RNA quantification was followed by cDNA conversion and miRNA or mRNA quantification by qRT-PCR performed in the real-time PCR system StepOnePlus (Applied Biosystems, Thermo Fischer Scientific, USA). For miRNA quantification, the miRCURY LNA universal RT miRNA PCR system (Exiqon, Denmark) was employed, in combination with pre-designed LNA primers (Exiqon, Denmark) (Table S1; Supplemental Materials and Methods), and the mouse small nucleolar RNA SNORD 110 was used as a reference. For mRNA quantification, the iQ SYBR Green supermix kit (Bio-Rad, USA) was employed, in combination with pre-designed primers (GeneGlobe, QIAGEN), and the hypoxanthine phosphoribosyltransferase (HPRT) mRNA was used as reference. miRNA and mRNA fold changes were determined by the Pfaffl method. Details of each protocol can be found in Supplemental Materials and Methods.

Barros-Viegas A.T., Carmona V., Ferreiro E., Guedes J., Cardoso A.M., Cunha P., Pereira de Almeida L., Resende de Oliveira C., Pedro de Magalhães J., Peça J, & Cardoso A.L. (2020). miRNA-31 Improves Cognition and Abolishes Amyloid-β Pathology by Targeting APP and BACE1 in an Animal Model of Alzheimer’s Disease. Molecular Therapy. Nucleic Acids, 19, 1219-1236.

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Quantitative miRNA and mRNA Analysis

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RNA was isolated with the miRVana kit (Ambion, Gent, Belgium). We controlled its integrity and concentration using Nanodrop (ThermoScientific, Erembodegem, Belgium) and BioAnalyzer (Agilent, Diegem, Belgium). For murine samples, 1 μg of total RNA was reverse transcribed with the miScript cDNA synthesis kit (Qiagen, Venlo, the Netherlands); for human samples, 0.5 μg of total RNA was reverse transcribed. Real‐time quantitative PCR was performed with SYBR green mix (Applied Biosystems, Gent, Belgium) on an ABI Prism 7500 (Applied Biosystems). LNA primers (Exiqon, Vedbaek, Denmark) were used to detection mature miRs. U6 was used as an endogenous control for miRs; GAPDH was used as a housekeeping gene for mRNAs.

Van Aelst L.N., Summer G., Li S., Gupta S.K., Heggermont W., De Vusser K., Carai P., Naesens M., Van Cleemput J., Van de Werf F., Vanhaecke J., Thum T., Waer M., Papageorgiou A.‐., Schroen B, & Heymans S. (2015). RNA Profiling in Human and Murine Transplanted Hearts: Identification and Validation of Therapeutic Targets for Acute Cardiac and Renal Allograft Rejection. American Journal of Transplantation, 16(1), 99-110.

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Validating miRNA Expression Changes in Rat Irradiation

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Validation of changes in miRNA expression of selected markers was performed by RT-qPCR from the blood of rats used for miRNA-seq as well as from independently irradiated and control rats. Total RNA from each rat were required to meet the following quality criteria: 1) A_260/280 > 1.6, 2) yield of RNA was enough to carry out all the RT-qPCR reactions needed for all the primers tested. There was no pre-amplification step prior to PCR. LNA-primers were obtained from Exiqon and reactions were carried out after reverse transcription using miRCURY LNA^TM Universal RT microRNA PCR kit from Exiqon with sybr green mastermix from Biotool. The relative expression of each miRNA to a reference miR-191-5p was calculated by the formula: 2^−∆CT (∆C_T=C_T (target-reference). Data was then normalized to the mean relative expression of controls, to determine the fold change after radiation. MiR-191-5p was used as reference for normalization because it shows consistent read counts in the libraries by sequencing.

Gao F., Liu P., Narayanan J., Yang M., Fish B.L., Liu Y., Liang M., Jacobs E.R, & Medhora M. (2017). Changes in miRNA in the lung and whole blood after whole thorax irradiation in rats. Scientific Reports, 7, 44132.

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