Takara sybr primescript mirna rt pcr kit

Manufactured by Takara Bio

Sourced in Japan

The Takara SYBR® PrimeScript™ miRNA RT-PCR Kit is a laboratory tool designed for the detection and quantification of mature microRNA (miRNA) molecules. It provides a complete system for reverse transcription and real-time PCR amplification of miRNA targets.

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Lab products found in correlation

Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions) Abi prism 7500 sequence detection system, by Thermo Fisher Scientific (1 mentions) Abi viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions) First strand cdna synthesis kit, by GeneCopoeia (1 mentions) All in onetm qpcr mix, by GeneCopoeia (1 mentions) One step primescript mirna cdna synthesis kit, by Takara Bio (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

TaKaRa SYBR® PrimeScriptTM RT‐PCR Kit TaKaRa PrimeScript RT-PCR Kit SYBR PrimeScript RT-PCR Kit PrimeScript RT-PCR kit TAKARA PrimeScript Kit PrimeScript RT kit MiRNA RT Kit PrimeScript RT RT-PCR kit Takara kit

3 protocols using takara sybr primescript mirna rt pcr kit

Serum miRNA and MALAT1 detection

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An equal volume of serum (250 μL) was processed during RNA isolation for each subject. For detection of microRNAs (miRNAs), a TaKaRa SYBR PrimeScript™ miRNA RT-PCR kit (Takara, Japan) was used to reverse transcribe serum RNA into cDNA (Takara, Japan) according to the manufacturer’s protocol.
For the detection of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), reverse transcriptase M-MLV was used to reverse transcribe serum RNA into cDNA (Takara, Japan) (RNase^H−) according to the manufacturer’s protocol.
An ABI ViiA™ 7 real-time PCR system (Applied Biosystems) and a TaKaRa SYBR PrimeScript™ miRNA RT-PCR kit (Takara, Japan) were used to perform quantitative PCR. The universal primers were provided by the manufacturer. The miRNA-specific forward primers are shown in Supplementary Table 1.
The quantitative PCR reactions were conducted as follows: at 95°C for 30 seconds; at 95°C for 5 seconds; and at 60°C for 31 seconds, for 40 cycles. The expression of miRNA of all of the samples was normalized to cel-miR-39 [9 (link)]. GAPDH was used as a reference gene for the gene expression normalization [10 (link)]. All the reactions were conducted in triplicate, and the 2^−ΔΔCt method was used to calculate the relative expression levels of the target genes.

Huang S.K., Luo Q., Peng H., Li J., Zhao M., Wang J., Gu Y.Y., Li Y., Yuan P., Zhao G.H, & Huang C.Z. (2018). A Panel of Serum Noncoding RNAs for the Diagnosis and Monitoring of Response to Therapy in Patients with Breast Cancer. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 24, 2476-2488.

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Urine miRNA Extraction and qPCR Analysis

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Total RNA was isolated from urine supernatant (200 μl) using miRCURY™ RNA Isolation Kit-Biofluids (Exiqon, Vedbaek, Denmark) according to the manufacturer's instructions. Total RNA concentration was measured on a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA). First-strand cDNA was generated from about 100 ng of RNA using the Takara SYBR^® PrimeScript™ miRNA RT-PCR Kit (Takara Bio Inc) in a final volume of 20 μl reverse transcription (RT) reaction system by following the protocol provided by the manufacturer. The RT condition was set as follows: 37°C for 60 min, 85°C for 5 sec and 4°C for 60 min. cDNA synthesis was performed in triplicate to ensure enough quantity for qPCR assays. Then, 2 μl of 5-fold diluted cDNA was added in a qPCR reaction consisting of 12.5 μl SYBR Premix Ex Taq II, 0.5 μl Dye II 2 μl of 5 μM forward primer, 1 μl of 10 μM Uni-miR RT-qPCR Primer, and 7 μl of ddH₂O. The cycling conditions were 95°C for 30 sec, 45 cycles of 95°C for 5 sec and 57°C for 34 sec. All reactions in triplicate were assessed in the ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA). The relative expression fold change was calculated by using the 2^-ΔΔCt method [39 (link)].

Du L., Jiang X., Duan W., Wang R., Wang L., Zheng G., Yan K., Wang L., Li J., Zhang X., Pan H., Yang Y, & Wang C. (2017). Cell-free microRNA expression signatures in urine serve as novel noninvasive biomarkers for diagnosis and recurrence prediction of bladder cancer. Oncotarget, 8(25), 40832-40842.

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Hypoxia-Induced lncRNA and miRNA Expression

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After transfection and hypoxia exposure, the cells in 24-well plates were washed twice with ice-cold PBS. Total RNA in cell was extracted by using the Trizol reagent (Life Technologies Corporation, USA). The purity and concentration of RNA in the extracts was tested by UV spectrophotometry. To test the expression of lncRNA GAS5 and TP53INP1, First Strand cDNA Synthesis Kit (GeneCopoeia, USA) and 2xAllinOneTMQ-PCRMix (GeneCopoeia) were used. To test the expression of miR-378, miR-21, miR-30b, miR-93 and miR-142-5p, One Step PrimeScript® miRNA cDNA Synthesis Kit and Takara SYBR® PrimeScript™ miRNA RT-PCR Kit (both from TaKaRa Biotechnology, China) were used. β-actin was used as an internal control for lncRNA GAS5 and TP53INP1, and U6 for miRNAs. Fold changes were calculated by the 2^−ΔΔCt method.

Du J., Yang S.T., Liu J., Zhang K.X, & Leng J.Y. (2019). Silence of LncRNA GAS5 Protects Cardiomyocytes H9c2 against Hypoxic Injury via Sponging miR-142-5p. Molecules and Cells, 42(5), 397-405.

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