Total RNA was extracted from HCC cell lines and tissue samples using TRIZOL ® reagent (Takara, Otsu, Japan). For quantify miR-449a, cDNA was synthesized with the TaqMan MicroRNA reverse transcription kit (GenePharma, Jiangsu, China), and then was quantified using the QuantMir RT Kit (GenePharma, Jiangsu, China) under LightCycler® Instrument (Roche Diagnostics, Indiana, USA) with miR-449a (forward: 5ʹ-CTCGCTGGCAGTGTATTGTTAG-3ʹ;reverse:5ʹ-TATCGTTGTACTCCAGACCAAGAC-3ʹ) and U6 (forward: 5ʹ-CTCGCTTCGGCAGCACA-3ʹ; reverse: 5 ʹ-AACGCTTCACGAATTTGCGT-3ʹ). The expression of Notch1, RNA was reverse-transcribed into cDNA by a PrimscriptTM RT Reagent (Takara, Otsu, Japan), then was quantified using Real-time quantitative PCR Mixture Reagent (Takara, Otsu, Japan) under LightCycler® Instrument with Notch1 (forward: 5ʹ-CGGGTCCACCAGT TTGAATG-3ʹ; reverse: 5ʹ-GTTGTATTGGTTCGGCACCAT-3ʹ) and β-actin (forward: 5ʹ-CACCATGAAGATCAAGATCATTGC-3ʹ; reverse: 5ʹ-GGCCGGACTCATCGT ACTCCTGC-3‘). The relative expression of miR-449a was normalized to U6 and the expression of Notch1 was normalized to β-actin by using 2-△△Ct method.
Lightcycler instrument
Manufactured by Takara Bio
Sourced in Japan
The LightCycler® Instrument is a real-time PCR system designed for qualitative and quantitative nucleic acid analysis. It enables rapid and reliable detection and quantification of target sequences.
Automatically generated - may contain errors
Lab products found in correlation
Taqman microrna reverse transcription kit, by GenePharma (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Lightcycler instrument, by Roche (1 mentions)
Takara reverse transcriptase, by Takara Bio (1 mentions)
Sybr green 2 kit, by Takara Bio (1 mentions)
Trizol reagent, by Beyotime (1 mentions)
2 protocols using lightcycler instrument
1
Quantification of miR-449a and Notch1 in HCC
2
Quantitative Analysis of Retinal Gene Expression
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Total RNA was respectively extracted from the retinas in three subgroups (n = 6 per subgroup, every two eyeball specimens in each group were pooled) using trizol reagent (Beyotime, Biotechnology, China) according to the manufacturer’s protocol. This was followed by reverse-transcription of 1 μg of total RNA to cDNA using Takara reverse transcriptase and the random primer provided in the kit (Takara, Japan). QPCR was performed on a Roche LightCycler instrument with Takara SYBR green II kit using resultant cDNA as template. The primer sequences were listed in Table 1 . The protocol for the qPCR was an initial denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 59°C for 60 s, and extension at 97°C for 1 s. At the end of the amplification, calculating the relative expression of target genes in each group with the 2–△△ CT method (Ding et al., 2020 (link); Wu et al., 2020 (link)).
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