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Bulge loop mirna rt qpcr starter kit

Manufactured by RiboBio
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Sourced in China

The Bulge-Loop™ miRNA RT-qPCR Starter Kit is a laboratory equipment product designed for the detection and quantification of microRNA (miRNA) expression. The kit includes reagents and protocols necessary for reverse transcription and real-time quantitative PCR (RT-qPCR) analysis of miRNA targets.

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Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (4 mentions) Primescript rt reagent kit, by Takara Bio (2 mentions) Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions) Transscript 2 one step gdna removal and cdna synthesis supermix kit, by Transgene (1 mentions) Rneasy ffpe kit, by Qiagen (1 mentions) Transstart tip green qpcr supermix kit, by Transgene (1 mentions) Lightcycler 96 system, by Roche (1 mentions) Nanodrop 2000 instrument, by Thermo Fisher Scientific (1 mentions) Abi 7500 system, by Thermo Fisher Scientific (1 mentions) Mirna specific stem loop primer, by RiboBio (1 mentions) Sybr green master mix, by Vazyme (1 mentions) Reverse transcription kit, by Vazyme (1 mentions) Bulge loop mirna rt qpcr primer set, by RiboBio (1 mentions) Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions) Rnaiso plus reagent, by Takara Bio (1 mentions) Sybr premix ex taq 2 kit, by Takara Bio (1 mentions)

5 protocols using bulge loop mirna rt qpcr starter kit

1

Comprehensive RNA Extraction and qPCR Analysis

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Total RNAs from tissues and cells were extracted using Trizol reagent (Invitrogen). Total RNAs from FFPE were extracted using RNeasy®FFPE Kit (Qiagen, Germany). cDNA was synthesized with the TransScript® II One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen, Beijing, China) according to the manufacturer’s instructions. RT-qPCR was performed on the LightCycler®96 system (Roche) using a standard protocol from the TransStart® Tip Green qPCRSuperMix Kit (TransGen, Beijing, China). The results were normalized to GAPDH. The sequences of primers used for RT-qPCR assays are listed in Additional file 1.
For the detection of miRNA expression, reverse transcription and qPCR were performed using stem-loop primers and Bulge-Loop™ miRNA RT-qPCR Starter Kit from Ribobio (Guangzhou, China). U6 snoRNA was used as the endogenous control. All experiments were performed in triplicate according to the manufacturer’s manual. Expression fold changes were calculated using 2-∆∆Ct methods.
Dong S., Wang R., Wang H., Ding Q., Zhou X., Wang J., Zhang K., Long Y., Lu S., Hong T., Ren H., Wong K., Sheng X., Wang Y, & Zeng Y. (2019). HOXD-AS1 promotes the epithelial to mesenchymal transition of ovarian cancer cells by regulating miR-186-5p and PIK3R3. Journal of Experimental & Clinical Cancer Research : CR, 38, 110.
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2

RNA Extraction and RT-qPCR Analysis

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Total RNA was extracted from tissue and cells (1×106) with TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. The quantity and concentration of total RNA were determined by a NanoDrop 2000 instrument (Thermo Fisher Scientific, Inc.). RT-qPCR was performed as described previously (33 (link)). Briefly, total RNA was reverse transcribed to cDNA using a PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd.). qPCR was performed in a 96-well plate on an ABI 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with PowerUp SYBR-Green Master Mix (Thermo Fisher Scientific, Inc.) as per the procedure provided by the manufacturer. For detecting hsa-miR-23a-5p, a hsa-miR-23a-5p-specific stem-loop primer (Guangzhou RiboBio Co., Ltd.) was used for reverse transcription and RT-qPCR amplification was performed using the Bulge-Loop miRNA RT-qPCR Starter kit (Guangzhou RiboBio Co., Ltd.). The thermocycling conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. GAPDH (for circRNA and mRNA) or U6 (for miRNA) was used as reference control. Relative expression level was calculated by the 2−ΔΔCq method (34 (link)). Primer sequences are listed in Table II.
Yang J., Li Y., Yu Z., Zhou Y., Tu J., Lou J, & Wang Y. (2020). Circular RNA Circ100084 functions as sponge of miR-23a-5p to regulate IGF2 expression in hepatocellular carcinoma. Molecular Medicine Reports, 21(6), 2395-2404.
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3

Quantifying mRNA and miRNA Expression

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Total RNAs were isolated from cells or liver tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The concentration and purity of RNA were detected by Nanodrop spectrophotometer. cDNA was synthesized using Reverse Transcription Kit (Vazyme Biotech, Nanjing, China), and RT-qPCR was performed using SYBR Green Master Mix (Vazyme Biotech, Nanjing, China). The relative mRNA level was normalized to the GAPDH gene. 2−△△Ct relative quantitative method was used to evaluate gene expression. The primer sequences used in this study are listed in Supplementary Table 1.
For miRNA analysis, the first-strand cDNA was synthesized using reverse transcriptase with a miRNA-specific stem-loop primer (RiboBio, Guangzhou, China) and RT-qPCR analysis was employed using the Bulge-Loop miRNA RT-qPCR Starter Kit (RiboBio, Guangzhou, China). The relative expression level of miRNA was normalized to U6 gene by using the 2−△△Ct method.
Chen X., Zhang D., Wang Y., Chen K., Zhao L., Xu Y., Jiang H, & Wang S. (2020). Synergistic antifibrotic effects of miR-451 with miR-185 partly by co-targeting EphB2 on hepatic stellate cells. Cell Death & Disease, 11(5), 402.
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4

RNA Extraction and miRNA Quantification

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Total RNA was extracted from the cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol and assessed for quality by One-Drop (Eppendorf, Hamburg, Germany). Mature miRNAs were quantified using the Bulge-Loop miRNA RT-qPCR starter kit and the Bulge-Loop miRNA RT-qPCR primer set (RIBOBIO, Guangzhou, China). The 2-fold Ct method was used to calculate the fold change in gene expression. Primer sequences were shown in Table 1.
Tang L., Chen Y., Tang X., Wei D., Xu X, & Yan F. (2020). Long Noncoding RNA DCST1-AS1 Promotes Cell Proliferation and Metastasis in Triple-negative Breast Cancer by Forming a Positive Regulatory Loop with miR-873-5p and MYC. Journal of Cancer, 11(2), 311-323.
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5

Quantification of miR-503 Expression

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Total RNA was extracted from H9c2 cells or heart tissues using RNAiso Plus reagent according to the manufacturer's instructions (TakaRa, Japan) as described [36 (link)]. The whole heart tissue was ground into powder with liquid nitrogen, and about 50 mg powder was homogenized with RNAiso Plus reagent. cDNA was synthesized from 1 μg of total RNA from each sample with PrimeScript RT reagent kit (Takara) and Bulge-Loop™ miRNA RT-qPCR Starter Kit (RiboBio). The expression level of mature miR-503 was quantified by RT-qPCR using SYBR Premix Ex Taq II kit (Takara). U6 was regarded as an internal reference of miR-503. Amplification of cDNA was performed on a StepOnePlus Real-time PCR System (Applied. Biosystems). The reaction was initiated at 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. The 2-∆∆CT was used as relative quantification method.
He Y., Cai Y., Sun T., Zhang L., Irwin M.G., Xu A, & Xia Z. (2022). MicroRNA-503 Exacerbates Myocardial Ischemia/Reperfusion Injury via Inhibiting PI3K/Akt- and STAT3-Dependent Prosurvival Signaling Pathways. Oxidative Medicine and Cellular Longevity, 2022, 3449739.
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