Total RNA was extracted using the Trizol method (Takara, Japan). RNA concentration and purity were determined by Colibri Microvolume Spectrometer (Titertek-Berthold, Germany). This was followed by reverse transcription of 1 μg total RNA. PrimeScript RT Reagent Kit were purchased from Takara (Shanghai, China). PCR amplification was conducted in 10 μl total volume consisting of 3 μl cDNA, 5 μl SYBR green mixture, 1.6 μl H2O, and 0.4 μl of primer mix (10 μM). Primers used for quantitative RT-PCR or for cloning are listed in Supporting Table 1 . The amplification steps were 40 cycles of denaturation at 94 °C for 15 s, extension at 60 °C for 60 s, using the ABI ViiA 7Dx real-time PCR system (Life Technologies, NY, USA). Gene expression was normalized to 18S rRNA as internal control. For bulge-loop miRNA qRT-PCR, primer set specific for miR-942 was designed by RiboBio (Guangzhou, China). U6 small nucleolar RNA (RiboBio Co., Ltd) was used for miRNA normalization. qRT-PCR values were calculated by relative quantification using the delta-delta CT method.
U6 small nucleolar rna
Manufactured by RiboBio
Sourced in China
U6 small nucleolar RNA is a non-coding RNA molecule that plays a core role in the biogenesis and function of the spliceosome. It is essential for the proper splicing of pre-mRNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Stem loop primers, by RiboBio (2 mentions)
Trizol method, by Takara Bio (1 mentions)
Abi viia 7dx real time pcr system, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Iq5 system, by Bio-Rad (1 mentions)
Sybr green kit, by Takara Bio (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Lipofectamine ltx with plus reagent, by Thermo Fisher Scientific (1 mentions)
Mir 21 5p mimic, by RiboBio (1 mentions)
4 protocols using u6 small nucleolar rna
1
Comprehensive RNA Extraction and qRT-PCR Analysis
2
RNA Extraction and RT-qPCR Analysis
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Total RNA was extracted from the cells using TRIzol reagent (Takara, Kyoto, Japan). cDNA was synthesized and RT-qPCR reactions were performed in triplicate using the SYBR green kit (Takara) with a Bio-Rad iQ™5 system. microRNA assay was detected with stem-loop primers purchased from RiboBio. U6 small nucleolar RNA (Guangzhou RiboBio Co., LTD, Guangzhou, China) was used for the normalization. The primer sequences used for genes studied are listed in Table 1 . The final relative expression fold differences, with gene expression NC as a control, were calculated as 2−ΔΔCt for each gene.
3
MicroRNA Assay and Transfection in Cardiomyocytes
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MicroRNA assay was detected with stem loop primers purchased from Ribobio (Guangzhou RiboBio, Guangzhou, China). U6 small nucleolar RNA (Guangzhou RiboBio) was used for the normalization. The sequence of rno-mir-182 was 5′-ACGCGGGUCUAGCUGCCGGAGGCCUCCCACCGUUUUUGGCAAUGGUAGAACUCACACCGGUA-3′. Rno-mir-182 mimics (Guangzhou RiboBio) were transfected to rat neonatal cardiomyocytes with Lipofectamine 3000 (Invitrogen).
4
miR-21-5p Functional Assay Protocol
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Sequences of miR-21-5p were collected from miRBase. miR-21-5p mimic, inhibitor (antagomir-21) and their corresponding negative control were all obtained from Guangzhou RiboBio Co., Ltd.
U6 small nucleolar RNA (Guangzhou RiboBio Co., Ltd.) was used as the endogenous control. Plasmids were amplified and purified using the HiSpeed Plasmid Midi Kit (cat. no. 12643; Qiagen, Inc.) and stored in ddH2O at 1 µg/µl before use. All plasmids were transfected into cells using Lipofectamine® LTX with Plus™ reagent (Invitrogen; Thermo Fisher Scientific, Inc.).
U6 small nucleolar RNA (Guangzhou RiboBio Co., Ltd.) was used as the endogenous control. Plasmids were amplified and purified using the HiSpeed Plasmid Midi Kit (cat. no. 12643; Qiagen, Inc.) and stored in ddH2O at 1 µg/µl before use. All plasmids were transfected into cells using Lipofectamine® LTX with Plus™ reagent (Invitrogen; Thermo Fisher Scientific, Inc.).
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