Mirna specific primer

Manufactured by Thermo Fisher Scientific

Sourced in United States, Canada

The MiRNA-specific primers are a set of oligonucleotide sequences designed to target and amplify specific microRNA (miRNA) molecules. These primers are used in reverse transcription-polymerase chain reaction (RT-PCR) and other miRNA detection and quantification techniques, allowing for the sensitive and accurate measurement of miRNA expression levels in biological samples.

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29 protocols using mirna specific primer

Profiling miRNA and mRNA in Familial and Sporadic Breast Cancers

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MiRNA and mRNA expression analyses were performed on an independent set of breast tumors stratified into 19 familial (8 BRCA1/2-related and 11 BRCAX) and 10 sporadic BCs.
Total RNA was extracted from formalin-fixed, paraffin-embedded breast cancers, as described above. Briefly, for detection of miR-92a-1*, miR-1184 and miR-943 expression levels, 10 ng of total RNA were reverse transcribed using the TaqMan^® MicroRNA Reverse Transcription Kit and miRNA specific primers according to the manufacturer's protocol (Applied Biosystems). Real Time PCR analysis was performed on the ABI Prism 7000 Sequence Detection System (Applied Biosystems) using 3 μl of RT products in a reaction mixture containing TaqMan miRNA assay and the TaqMan Universal PCR Master Mix, according to the manufacturer's instructions (Applied Biosystems). All PCR reactions were performed in triplicate including no-template controls. Relative quantities of each miRNA were calculated using the ΔΔCt method after normalization with endogenous reference RNU 48.

Danza K., De Summa S., Pinto R., Pilato B., Palumbo O., Carella M., Popescu O., Digennaro M., Lacalamita R, & Tommasi S. (2017). TGFbeta and miRNA regulation in familial and sporadic breast cancer. Oncotarget, 8(31), 50715-50723.

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Comparative miRNA Expression Analysis

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Using the same total RNA samples, relative expression levels of the selected miRNAs (mmu-miR-3078-5p, mmu-miR-200c-3p, mmu-miR-496a-5p, mmu-miR-412-5p, mmu-miR-323-5p) were analysed after reverse transcription with specific RT primers (TaqMan® MicroRNA Assay, Applied Biosystems, Cheshire, UK) following the manufacturer’s guidelines. Fifty nanograms of the total RNA from the samples was used for the reverse transcription. The cDNA was amplified by the qRT-PCR with Universal TaqMan Mix (with no Amperase Ung) and miRNA-specific primers (Applied Biosystems), following the manufacturer’s protocol. Reactions were performed on the qRT-PCR machine, CFX connect (BioRad, Reinach, Switzerland). All reactions were performed in triplicate, and their relative abundances were analysed by normalising with respect to small nucleolar RNA (snoRNA). Two groups were compared according to their 2^−ddCt values, and the significance was calculated using a one-way analysis of variance (ANOVA), followed by Dunnett’s post hoc tests with the level of statistical significance set at P < 0.05.

Lee S., Woo J., Kim Y.S, & Im H.I. (2015). Integrated miRNA-mRNA analysis in the habenula nuclei of mice intravenously self-administering nicotine. Scientific Reports, 5, 12909.

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Quantitative Analysis of miRNA

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RNA was extracted from PFC and hippocampus samples using miRNeasy mini kit (QIAGEN) following the manufacturer’s protocol. Reverse transcription was performed using miRNA-specific primers (Applied Biosystems, Foster City, CA, USA). The resulting cDNA was diluted 1:2 prior to performing quantitative real-time PCR (qPCR). qPCR was performed in triplicate using Taqman microRNA probes (Applied Biosystems Inc, CA, USA) following the manufacturer’s protocol.

Huang C., Wang Y., Wu Z., Xu J., Zhou L., Wang D., Yang L., Zhu B., Chen G., Liu C, & Yang C. (2021). miR-98-5p plays a critical role in depression and antidepressant effect of ketamine. Translational Psychiatry, 11, 454.

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Quantitative Real-Time PCR Workflow

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For quantitative real-time PCR (qPCR), 1.0 μg of total RNA per sample was used for reverse transcription to cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). cDNA was diluted 1:10 in ultrapure water and 5 μL of this dilution was used per reaction. qPCR was performed using TaqMan Fast Advanced Master Mix and species-specific TaqMan primers. The expression of the gene B2M (beta-2 microglobulin) was used as internal control. For miRNA qPCR, 350 ng of total RNA per sample was used for reverse transcription to cDNA using TaqMan microRNA Reverse Transcription Kit and miRNA-specific primers (Applied Biosystems), and 0.16 μL of cDNA was used per reaction. microRNA qPCR was performed using TaqMan Universal Master Mix II, no UNG, and species-specific TaqMan miRNA primers, using U6 as internal control [29 (link)]. qPCR reactions were run in duplicate in a StepOnePlus Real-Time PCR system (Applied Biosystems) and ΔCt calculations were made using StepOne software (Applied Biosystems).

Kim T., Valera E, & Desplats P. (2019). Alterations in Striatal microRNA-mRNA Networks Contribute to Neuroinflammation in Multiple System Atrophy. Molecular neurobiology, 56(10), 7003-7021.

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Quantification of miRNA Expression

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Total RNA, including miRNA, was isolated using the miRNeasy Mini kit and treated with DNase I (Qiagen, Cat# 79254). For quantitative analysis of miRNA expression, 80 ng of total RNA was reverse-transcribed using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA; Cat# 4366597) and miRNA-specific primers (Applied Biosystems), and PCR was performed using TaqMan Fast Advanced Master Mix and TaqMan microRNA assays (Applied Biosystems, Cat# 4444558). To measure primary miRNA levels, total RNA was reverse-transcribed with SuperScript VILO MasterMix (Invitrogen, Cat# 11755-250), and synthesized cDNA was quantified using TaqMan Fast Advanced Master Mix and TaqMan Pri-miRNA Assays (Applied Biosystems). RNU48 small nuclear RNA, TBP mRNA (TATA box–binding protein), or PPIA mRNA (peptidylprolyl isomerase A) was used as an internal control. Relative expression levels were calculated by the comparative cycle threshold (Ct) method. All PCRs were performed in triplicate on an Applied Biosystems ViiA7 Real-Time PCR System.

Fujiwara Y., Saito M., Robles A.I., Nishida M., Takeshita F., Watanabe M., Ochiya T., Yokota J., Kohno T., Harris C.C, & Tsuchiya N. (2018). A Nucleolar Stress–Specific p53–miR-101 Molecular Circuit Functions as an Intrinsic Tumor-Suppressor Network. EBioMedicine, 33, 33-48.

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Quantitative Analysis of mRNA and miRNA Expression

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Cells were washed twice with PBS, and total RNA was isolated using TRI‐Reagent (Sigma, USA) according to manufacturer's instructions. For mRNA, cDNA was prepared by reverse transcription‐PCR (RT‐PCR) using the high‐capacity cDNA synthesis kit (Applied Biosystems, USA) using the following program: 1 hour at 37°C and 5 minutes at 95°C. Quantitative RT‐PCR (qRT‐PCR) was performed with KAPA SYBR FAST Universal kit (KAPA Biosystems, USA) using the appropriate specific primers (listed in Supporting Information Table S2) as follows: 3 minutes at 95°C, 40 cycles of 5 seconds at 95°C, 20 seconds at 60°C, and 10 seconds at 72°C. For TaqMan assays of microRNAs (miRNAs), 5 μl RNA (5 ng/μl) was subjected to RT‐PCR using the reverse transcription kit and miRNA‐specific primers (Applied Biosystems, USA) followed by qRT‐PCR using TaqMan universal master mix and TaqMan miRNA‐probes or U54 as control (Applied Biosystems). The relative amounts of each mRNA or miRNA were normalized to glyceraldehyde‐3‐phosphate dehydrogenase or U54, respectively, and the relative expression of each reaction was calculated as a fold change relative to the control sample. Samples were cycled using Applied Biosystems StepOnePlus RT‐PCR system.

Bhattacharya S., Serror L., Nir E., Dhiraj D., Altshuler A., Khreish M., Tiosano B., Hasson P., Panman L., Luxenburg C., Aberdam D, & Shalom‐Feuerstein R. (2019). SOX2 Regulates P63 and Stem/Progenitor Cell State in the Corneal Epithelium. Stem Cells (Dayton, Ohio), 37(3), 417-429.

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Quantification of miRNA by TaqMan and SYBR Green qPCR

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Taqman MicroRNA Assays were used for miRNA quantification. In brief, 2 μl of RNA was reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit and miRNA-specific primers (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions in a reaction volume of 15 μl. qPCR was done using the FAST chemistry (Applied Biosystems) with the manufacturer provided miRNA-specific assays in ABI PRISM 7900 HT Real-Time PCR system (Applied Biosystems). The assays analysed are listed in Table 1.
miR-29a and miR-92 were tested using the Qiagen technology as reported by Huang et al (2010) (link) and Ng et al (2009) (link). Briefly, 4 μl of RNA was reverse transcribed to cDNA using the miScript Reverse Transcription Kit (Qiagen) according to the manufacturer's instructions in a reaction volume of 20 μl. SYBR Green chemistry (Qiagen) was used to perform the qPCR with the manufacturer provided miScript Universal Primer and miScript Primer Assay in LightCycler 480 II Roche.

Zanutto S., Pizzamiglio S., Ghilotti M., Bertan C., Ravagnani F., Perrone F., Leo E., Pilotti S., Verderio P., Gariboldi M, & Pierotti M.A. (2014). Circulating miR-378 in plasma: a reliable, haemolysis-independent biomarker for colorectal cancer. British Journal of Cancer, 110(4), 1001-1007.

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Quantitative Analysis of miRNA and Gene Expression

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For miRNA first strand DNA (cDNA) synthesis, miRNA specific primers purchased from Applied Biosystems and “TaqMan MicroRNA reverse transcription Kit (Applied Biosystems) were used to reverse transcribe equal amounts of total RNA. For miRNA expression analysis, TaqMan Fast Advanced Master Mix (Applied Biosystems) was used and microRNA specific probes were purchased from Applied Biosystems. MiRNA expression data were normalized to RNU43.
cDNA synthesis was performed with “amfiRivert cDNA Synthesis Platinum Master Mix” (GenDepot) according to the manufacturers' instructions. For gene expression analysis, SYBR Green PCR Master Mix of Applied Biosystems was used. Expression data were normalized to β-actin. Primer sequences used for Quantitative real time PCR (Q-RT-PCR) are provided in Supplementary Table 2. Q-RT-PCR was carried out in a StepOnePlus™ real-time thermal cycler (Applied Biosystem) using standard parameters. Each experiment was performed in triplicate and the differences in expression levels were evaluated using 2^{− ΔΔCT} method.

Karatas O.F., Wang J., Shao L., Ozen M., Zhang Y., Creighton C.J, & Ittmann M. (2017). miR-33a is a tumor suppressor microRNA that is decreased in prostate cancer. Oncotarget, 8(36), 60243-60256.

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Reverse Transcription and qPCR Analysis

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Complementary DNA was synthesized from 300 ng RNA using SuperScript II reverse transcriptase (Invitrogen, Paisley, UK) and either random hexamers or miRNA-specific primers (Applied Biosystems, Paisley, UK) according to the manufacturer’s instructions. 18S rRNA was used as the housekeeping gene. Complementary DNA was stored at −20 °C. The relative quantitation of gene expression was performed using an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Paisley, UK), following the manufacturer’s protocol.

Swingler T.E., Niu L., Pontifex M.G., Vauzour D, & Clark I.M. (2022). The microRNA-455 Null Mouse Has Memory Deficit and Increased Anxiety, Targeting Key Genes Involved in Alzheimer’s Disease. International Journal of Molecular Sciences, 23(1), 554.

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Transcriptomic Analysis of PDAC Samples

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TarBase v.6 [11 ] and MiRWalk v.2 [12 (link)] database were used to provide information about validated or predictive miRNAs-genes target interaction.
Total RNA was extracted from 2μm formalin-fixed, paraffin-embedded tissues sections using the RNeasy^® FFPE Kit (QIAGEN). For transcripts level analysis, 500 ng of total RNA were reverse transcribed using the High Capacity cDNA Reverse Transcription Kit, according to the manufacturer's protocol (Applied Biosystem). The ID assays used were the following: human VEGFA (Hs00900055_m1), human HIF1A (Hs00153153_m1), human Ang-1 (Hs00375822_m1), human Tie-2 (Hs00176096_m1) and human Ang-2 (Hs01048042_m1). RN18S1 (Hs03928985_g1) was used as the endogenous reference.
Briefly, for detection of miRNAs expression levels, 10 ng of total RNA were reverse transcribed using the TaqMan^® MicroRNA Reverse Transcription Kit and miRNA specific primers according to the manufacturer's protocol (Applied Biosystems), as previously described [36 (link)].
Quantitative real-time PCR was performed on the ABI Prism 7000 Sequence Detection System (Applied Biosystems) in accordance to the manufacturer's instructions (Applied Biosystems). MiRNAs and genes expression levels were calculated using ΔΔCt method after normalization with endogenous reference and PDAC as calibrator. All PCRs were performed in triplicate including no-template controls.

Silvestris N., Danza K., Longo V., Brunetti O., Fucci L., Argentiero A., Calabrese A., Cataldo I., Tamma R., Ribatti D, & Tommasi S. (2017). Angiogenesis in adenosquamous cancer of pancreas. Oncotarget, 8(56), 95773-95779.

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