For evaluating the feasibility of miR-21 inhibition by LNA-anti-miR, reverse transcriptase (RT)-PCR of microRNAs was carried out. After a day of transfection, FavorPrep™ Blood/Cultured Cell Total RNA Kit (Favorgen Biotech Co., Australia) was used based on column chromatography for the extraction of total RNA from the B16F10 cell line. A Nanodrop Epoch (BioTek, USA) was also used to measure the concentration of total RNA. Also, to synthesize cDNA from miRNA, cDNA synthesis kit (Exiqon, Denmark) was employed. To enhance cDNAs from miRNAs and detect miR-21 inhibition, BIOFACT™ 2X PCR master mix (BioFACT™, High ROX, Korea) was used in real-time PCR, along with miR-21-5P and miR-16 specific primers for normalization of real-time PCR; Exiqon (Denmark) provided all primers. Moreover, for 40-cycle real-time PCR, we used StepOnePlus real-time PCR system (Applied Biosystems, USA) was used. Finally, the relative miR21 expression was measured based on 2−ΔΔCT method.
Favorprep blood cultured cell total rna kit
Manufactured by Favorgen Biotech
Sourced in Australia
The FavorPrep™ Blood/Cultured Cell Total RNA Kit is a product designed to efficiently extract total RNA from blood samples and cultured cells. The kit utilizes a silica-membrane-based method to capture and purify the RNA, providing a reliable and consistent way to obtain high-quality RNA for various downstream applications.
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Lab products found in correlation
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Cdna synthesis kit, by Qiagen (1 mentions)
Mic qpcr cycler, by Bio Molecular Systems (1 mentions)
Luna universal qpcr master mix, by New England Biolabs (1 mentions)
Dnase free rnase, by Merck Group (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
2 protocols using favorprep blood cultured cell total rna kit
1
Evaluating miR-21 Inhibition by LNA-anti-miR
2
RNA Extraction and RT-qPCR Analysis
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Total RNA was extracted from YD-9 cells using a FavorPrep™ Blood/Cultured Cell Total RNA Kit (Favorgen Biotech Corporation), according to the manufacturer's instructions. A total of 300 ng total RNA was treated with RNase-free DNase (Sigma-Aldrich; Merck KGaA) for 15 min, followed by inactivation of DNase with EDTA treatment and heating. Total RNA was reverse-transcribed into cDNA using the First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. RT-qPCR was performed on cDNA samples with the Luna® Universal qPCR Master Mix (New England BioLabs, Inc.) using the Mic qPCR Cycler (Bio Molecular Systems). The thermocycling conditions were as follows: Initial denaturation at 95°C for 1 min was followed by cycles comprising denaturation at 95°C for 15 sec, annealing and extension at 60°C for 30 sec. The relative mRNA expression was calculated via the 2−ΔΔCq method (16 (link)). The sequences of the forward and reverse primers are presented in Table I .
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