The total RNA from cultured cells was extracted with NucleoZOL RNA Isolation kits (Takara Bio USA, Mountain View, CA) according to the manufacturer’s instructions.22–24 (link) The tissues were ground in an autoclaved grinding bowl with liquid nitrogen. The tissues were then treated using the total cellular RNA extraction protocol. Briefly, 20 mg of clinical tissue specimen were ground with liquid nitrogen and lysed with 500 µL NucleoZOL for 60 min at 4°C. Then, 200 µL RNase-free ddH2O was added to the lysates, and the mixture was vortexed vigorously. Subsequently, 500 µL of the supernatant was aliquoted into a new tube and precipitated with 500 µL isopropanol. After washing the resulting pellet twice with 75% ethanol, the total RNA was dissolved in 30 µL RNase-free ddH2O.
Nucleozol rna isolation kit
Manufactured by Takara Bio
Sourced in United States
NucleoZOL RNA isolation kit is a product from Takara Bio designed for the extraction and purification of total RNA from a variety of biological samples. It utilizes a specialized reagent for efficient lysis and denaturation of samples to release RNA, which is then selectively isolated and purified.
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5 protocols using nucleozol rna isolation kit
1
Total RNA Extraction from Tissue
2
Total RNA Extraction from Tissues
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The cultured cell total RNA was extracted with NucleoZOL RNA Isolation kits (Takara Bio USA, Mountain View, CA) according to the manufacturer's instructions as described [16] [17] [18] .
The tissues were grinded in the autoclaved grinding bowl with liquid nitrogen. The tissues were then treated as the cultured cells, following the cell total RNA extraction protocol.
Briefly, 20 mg of clinical tissues was grinded with the liquid nitrogen and lysed with 500 µL NucleoZOL for 60 mins at 4°C. 200 µL RNase free ddH2O was added to the lysate and vortexed vigorously. Then, 500 µL of the supernatant was pipetted and precipitated with 500 µL isopropanol. After washing with 75% ethanol twice, the total RNA was dissolved in 30 µL RNase free ddH2O.
The tissues were grinded in the autoclaved grinding bowl with liquid nitrogen. The tissues were then treated as the cultured cells, following the cell total RNA extraction protocol.
Briefly, 20 mg of clinical tissues was grinded with the liquid nitrogen and lysed with 500 µL NucleoZOL for 60 mins at 4°C. 200 µL RNase free ddH2O was added to the lysate and vortexed vigorously. Then, 500 µL of the supernatant was pipetted and precipitated with 500 µL isopropanol. After washing with 75% ethanol twice, the total RNA was dissolved in 30 µL RNase free ddH2O.
3
qRT-PCR Transcript Quantification Protocol
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Total RNA was purified by using the NucleoZOL RNA isolation kit (Takara Bio USA, Mountain View, CA, USA) and subjected to reverse transcriptase (RT) reactions using hexamer and Moloney murine leukemia virus (M-MuLV) RT (NEB). The resultant RT cDNA products were properly diluted and used as touchdown quantitative real-time PCR templates. Touchdown quantitative real-time PCR primers were designed with the Primer3 Plus program.70 (link) Touchdown quantitative real-time PCR reactions were carried out as described.21 (link),71 (link), 72 (link), 73 (link), 74 (link). Briefly, the SYBR Green (Bimake, Houston, TX, USA) qPCR reactions were carried out by using the following cycling program: 95°C for 3 min for 1 cycle; 95°C for 20 s, 66°C for 10 s per cycle, and then −3°C per cycle for four cycles; followed by 95°C for 10 s, 55°C for 15 s, and 70°C for 1 s for 40 cycles. Gapdh was used as a reference gene. The gene expression calculations were performed using the 2−ΔΔCt method.37 (link),56 (link),57 (link) Touchdown quantitative real-time PCR primer sequences are listed in Table S1 .
4
RNA Isolation and Quality Assessment
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Exponentially growing MCF7 or iMEF cells, or SASJ1 cells treated with or without Nutlin3A were subjected to total RNA isolation by using NucleoZOL RNA Isolation kit (Takara Bio USA, Mountain View, CA) according to the manufacturer’s instructions as described [15] (link), [16] . RNA integrity and quantity were assessed with an Agilent 2100 Bioanayzer (Santa Clara, CA). Briefly, RNA samples (1.0 µl) were loaded onto the Bioanalyzer RNA Nano Chips, along with size marker and subjected to electrophoresis according to the manufacturer’s instructions. Both gel images and electropherograms were obtained to assess the integrity and quantity of RNA samples. For quick and effective assessment of the quality of total RNA, we also ran 1% agarose gel with 1% bleach as previously described [17] (link).
5
Isolation and Purification of Small RNA
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Total RNA was isolated from exponentially growing HEK-293 cells using the NucleoZOL RNA Isolation Kit (Takara Bio USA, Mountain View, CA) according to the manufacturer’s instructions as described (20–22 (link)). To purify sRNA (<200 nt), we performed magnetic bead-based size selection with the commercially available Mag-Bind® TotalPure NGS magnetic beads (Omega Bio-Tek, Inc., Norcross, GA) as described previously (23 (link)). Briefly, 5 μg of total RNA was dissolved in 20 μl RNase-free molecular biology grade ddH2O and mixed with 20 μl Mag-Bind beads (i.e. RNA/magnetic bead vol/vol ratio of 1:1). The RNA/magnetic bead mixture was incubated at room temperature for 10 min. The mixture was subjected to a magnet, and the sRNA (<200 nt)-containing supernatant was collected, while the large transcripts (>200 nt) bound to beads and were discarded. The collected sRNA was subjected to PC8 phenol/chloroform extraction, followed by ethanol precipitation. The recovered sRNA was dissolved in 20 μl RNase-free molecular biology grade ddH2O for reverse transcription reactions, or kept at −80°C.
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