Total RNA was extracted from exosome with TRIzol reagent (Invitrogen, California, USA). The measurements were assessed with ΔCT which was normalized by U6 snRNA (RiboBio, Guangzhou, China). The expression levels of miR-483-5p, miR-423-5p, and miR-342-5p were measured via qRT-PCR using a Bulge-Loop™ miRNA reverse transcription kit (RiboBio, Guangzhou, China), and a Bulge-Loop™ miRNA qRT-PCR starter kit (RiboBio, Guangzhou, China). U6 snRNA (RiboBio, Guangzhou, China) was used as an endogenous control to quantify and normalize the results. The value 2 −ΔΔCT was used for comparative quantitation. All qRT-PCR reactions were performed in triplicate.
U6 snrna
Manufactured by RiboBio
Sourced in China
U6 snRNA is a small nuclear RNA (snRNA) component of the spliceosome, a complex of small nuclear ribonucleoproteins (snRNPs) and associated proteins that catalyzes the removal of introns from precursor messenger RNA (pre-mRNA). The U6 snRNA plays a crucial role in the splicing process by participating in the recognition and positioning of the splice sites.
Automatically generated - may contain errors
Lab products found in correlation
Bulge loop mirna qrt pcr starter kit, by RiboBio (3 mentions)
Rnaiso plus, by Takara Bio (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Tb green premix ex taq 2, by Takara Bio (1 mentions)
Sybr premix ex taq kit, by Takara Bio (1 mentions)
Abi prism 7500, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcriptase, by Takara Bio (1 mentions)
Stem loop rt primer, by RiboBio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Sybr green master mix, by Bio-Rad (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Qrt pcr system, by Thermo Fisher Scientific (1 mentions)
Quantstudio real time pcr system, by Vazyme (1 mentions)
Reverse transcription kit, by Vazyme (1 mentions)
Taq pro universal sybr qpcr master mix, by Vazyme (1 mentions)
6 protocols using u6 snrna
1
Exosomal miRNA Expression Analysis
2
Gastric Tissue and Cell RNA Profiling
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TRIzol® reagent (Takara Biotechnology Co., Ltd.) was used to extract total RNA from gastric tissues and cells. cDNAs were generated using the PrimeScript™ RT reagent kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions. SYBR® Premix Ex Taq (Takara Biotechnology Co., Ltd.) was used for qPCR according to the manufacturer's instructions. The thermocycling conditions were as follows: 95°C for 10 sec, followed by 40 cycles of 95°C for 5 sec, 60°C for 15 sec, and 72°C for 30 sec, the final extension was 72°C for 5 min. Primer sequences used for qPCR were as follows: MALAT1 forward, 5′-AGCGGAAGAACGAATGTAAC-3′ and reverse, 5′-GAACAGAAGGAAGAGCCAAG-3′; LC3B forward, 5′-GATGTCCGACTTATTCGAGAGC-3′ and reverse, 5′-TTGAGCTGTAAGCGCCTTCTA-3′; TRPM3 forward, 5′-ATACCCAGCACCAAAGACC-3′ and reverse 5′-TCTGAAGCACGGAGATACTG-3′; and GAPDH forward, 5′-TGAACGGGAAGCTCACTGG-3′ and reverse, 5′-TCCACCACCCTGTTGCTGTA-3′. The relative expressions levels were normalized to the endogenous control GAPDH and calculated using the 2−ΔΔCq method (39 (link)).
To detect miRNA-204, reverse transcription and qPCR were performed using a Bulge-Loop™ miRNA qPCR Primer Set for hsa-miR-204 (Guangzhou RiboBio Co., Ltd.) and U6 snRNA (Guangzhou RiboBio Co., Ltd.) according to the manufacturer's instructions and as previously described (40 (link)). U6 served as an internal control.
To detect miRNA-204, reverse transcription and qPCR were performed using a Bulge-Loop™ miRNA qPCR Primer Set for hsa-miR-204 (Guangzhou RiboBio Co., Ltd.) and U6 snRNA (Guangzhou RiboBio Co., Ltd.) according to the manufacturer's instructions and as previously described (40 (link)). U6 served as an internal control.
3
Quantifying mRNA and miRNA Expression in Mouse Lungs
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Total RNA was extracted from the lung tissues of mice using RNAiso Plus (Takara) and reverse-transcribed into complementary DNA using PrimeScript™ RT Master Mix (Takara). mRNA expression levels were quantified using TB Green® Premix Ex Taq™ II (Takara), with GAPDH expression serving as an internal control. Primers with the following sequences were used for real-time PCR: Shc4: forward: 5′-AGC CCA TAC TGG TGC CAT TGA-3′; reverse: 5′-GTT GAA CCA TTG TCC GGT GTG TAG-3′; Gria1: forward: 5′-AGC GGA CAA CCA CCA TCT CTG-3′; reverse: 5′-AAG GGT CGA TTC TGG GAT GTT TC -3′; and GAPDH: forward: 5′-TGC ACC ACC AAC TGC TTA G-3′; reverse: 5′-GGA TGC AGG GAT GAT GTT C-3′. The mir-7b and mir-486b primers and U6 snRNA (internal control) were purchased from RiboBio (Guangzhou, China). miRNA real-time PCR was performed by using the Bulge-loop™ miRNA qRT–PCR Starter Kit (RiboBio, Guangzhou, China) according to the manufacturer's protocol. Data were quantified using the comparative 2 − ΔΔCt method.
4
qRT-PCR Analysis of HIF-1α and miR-199a
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Total RNA was extracted using Trizol, and reverse-transcribed into cDNA with the Reverse Transcriptase M-MLV (TaKaRa). Real time PCR was performed using a SYBR Premix Ex Taq™ kit (TaKaRa) on the ABI PRISM 7500 sequence detection system (Applied Biosystems, Foster City, USA). In each assay, 1 μl of cDNA was used for amplification. The reactions were incubated in a 96-well optical plate at 95°C for 2 min, followed by 40 cycles of 95°C for 15 s and 60°C for 20 s. PCR primers used were as follows: HIF-1α F, 5′-GCA AGC CCT GAA AGC G-3′ and R, 5′-GGC TGT CCG ACT TTG A-3′; β-actin F, 5′-CAG AGC AAG AGA GGC ATC C-3′ and R, 5′-CTG GGG TGT TGA AGG TCT C-3′. For analysis of miRNA expression by qRT-PCR, reverse transcription and PCR were carried out using Bulge-Loop TM miRNA qPCR Primer Set for hsa-miR-199a (RiboBio, MQP-0101), and U6 snRNA (RiboBio, MQP-0201) according to the manufacturer's instructions. Expression of HIF-1α, relative to β-actin and miR-199a, relative to U6, was determined using the 2−ΔΔCTmethod. All qRT-PCRs were performed in triplicate, and the data are presented as means ± standard errors of the means (SEM).
5
Detecting miRNA and mRNA expression
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For miRNA or mRNA content detection, total RNA was extracted from cells or tissues using Trizol (Invitrogen, USA) according to the manufacturer’s instructions. cDNA was synthesized via RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). The reverse transcription reaction for miR-150 was carried out with stem-loop RT primers (RiboBio, China). QRT–PCR was performed using a SYBR Green Master Mix (Bio-Rad, USA). Relative expression was determined using U6 snRNA (primers from RiboBio, China) as an internal control for miR-150 and GAPDH as an internal control for FOXO4. The primers used are listed in Supplementary Table 1 .
6
Quantitative Analysis of Gene Expression
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Total RNA was extracted from the spinal cord neurons (1 × 106 cells/1 ml) and tissues (100 mg/1 ml) using RNAiso™ Plus (Takara, Japan) and 1 µg of total RNA was used to synthesize cDNA. The reverse transcription kit (Vazyme, China) was utilized for synthesizing cDNA under the following conditions: 15 min at 50°C, followed by 85°C for 5 s. The expression of total cDNA was measured using qRT-PCR with Taq Pro Universal SYBR qPCR Master Mix (Vazyme, China) and a qRT-PCR system (Thermo Fisher Scientific, DE); the total volume of q-PCR was 20 µl, including 10 µl of 2 × Taq Pro Universal SYBR qPCR Master Mix (Vazyme, China), 0.4 µl of each primer and 2 µl of cDNA diluted 5-fold; q-PCR was performed in a QuantStudio™ Real-Time PCR system with the following thermal cycling conditions: 30 s at 95°C followed by 40 cycles of 1 s at 95°C and 30 s at 60°C, and 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. The β-actin gene was used as the endogenous reference. For miRNA analysis, cDNA was prepared using a Bulge-Loop miRNA qRT-PCR Starter Kit (RiboBio, China), with U6 snRNA (RiboBio, China) being the endogenous reference. The 2-ΔΔCT approach was used to assess relative gene expression. The primer sets for Smo, xCT, GPX4, ACSL4, miR-6315, and β-actin genes are listed in Table 1 .
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