Total RNAs were extracted using Trizol (ThermoFisher Scientific) or NucleoZOL (TaKaRa) according to the manufacturer’s protocol. Extracted RNA was dissolved in RNase- and DNase-treated water and then immediately reverse-transcribed using HiScript Q RT SuperMix for qPCR (Vazyme). cDNA was stored at −20°C for subsequent qPCR analyses.
Nucleozol
Manufactured by Takara Bio
Sourced in United States
NucleoZOL is a reagent used for the isolation of high-quality total RNA from a variety of biological samples, including cells, tissues, and microorganisms. It is a mono-phasic solution containing phenol and guanidine isothiocyanate, which effectively lyses cells and denatures RNases to preserve the integrity of the extracted RNA.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (4 mentions)
Hiscript q rt supermix for qpcr, by Vazyme (2 mentions)
Sybr green qpcr reactions, by Selleck Chemicals (2 mentions)
M mulv reverse transcriptase, by New England Biolabs (2 mentions)
Cfx96 real time pcr, by Bio-Rad (2 mentions)
Cfx384 real time system, by Bio-Rad (1 mentions)
Luna universal one step rt qpcr kit, by New England Biolabs (1 mentions)
Dnase 1, by Takara Bio (1 mentions)
Nucleozol, by Macherey-Nagel (1 mentions)
Revertaid reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Veriti thermocycler, by Thermo Fisher Scientific (1 mentions)
Gotaq qpcr master mix, by Promega (1 mentions)
Accuscript high fidelity cdna synthesis kit, by Agilent Technologies (1 mentions)
Brilliant 3 ultra fast sybr green qpcr master mix, by Agilent Technologies (1 mentions)
Nucleospin rna clean up xs, by Takara Bio (1 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Facsaria fusion, by BD (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Quantstudio 12k flex, by Thermo Fisher Scientific (1 mentions)
Tb green premix ex taq 2 tli rnaseh plus, by Takara Bio (1 mentions)
14 protocols using nucleozol
1
Total RNA Extraction and cDNA Synthesis
2
Quantitative Analysis of Gene Expression
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Total RNA was isolated from cell lysates using NucleoZol (TaKaRa Bio) according to manufacturer’s instructions, and 1 μg of total RNA was suspended in 10 μl of RNase-free water. RT-qPCR was performed with 1 μl of 1:10 dilution of the RNA using Luna Universal One-Step RT-qPCR kit (New England BioLabs), with a total reaction volume of 10 μl in a Bio-Rad CFX384 Real-Time System (Bio-Rad). Primers used for Ago2 [90 (link)], Dicer [72 (link)], GPRC5A [62 (link)], SUMO3 [91 (link)] and SUMO1, SUMO2, and β-actin [23 (link)] transcripts are as previously described. The relative mRNA expression was derived from 2-ΔΔCT by use of the comparative threshold cycle (CT) method. The abundance of mRNA in each sample was normalized to the amount of actin mRNA.
3
Optimized RT-qPCR Protocol for Gene Expression
4
Ecdysone Reporter Assay in Hi5 Cells
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Hi5 cells (500,000 cells per well in a 24-well plate) were transfected with ecdysone reporter genes pERE-Luc (150 ng) and pERE-GFP (50 ng) together with RNA of infected cells (up to 500 ng) (31 (link)). One day after transfection, tebufenozide (ecdysone agonist) was added at 200 nM to induce reporter expression and cells were collected at 48 hrs for GFP (normalization) and luciferase (targeted by luciferase RNA hairpin) measurements. RNA was isolated from both soluble and insoluble cellular extracts by Nucleozol (Machery-Nagel, Germany). Total nucleic acid extracts were treated with RNase-free DNase I (1 U/μg for 15 min; TaKaRa) before Nucleozol extraction. As positive control, in vitro transcribed luciferase RNA hairpin was co-transfected at 1 to 10 ng quantities. Non-specific dsRNA (dsmalE) was used as negative control (31 (link)). GraphPad Prism 4 software was used for statistical analysis (unpaired t-test).
5
Fetuin-A Expression Analysis by qRT-PCR
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Total RNA was isolated from cells by NucleoZOL (Takara Bio USA, Inc., CA, USA) and converted into cDNA use of ToolsQuant II Fast RT kit (BIOTOOLS Co., Taipei, Taiwan). cDNAs were then used as templates for qRT-PCR. qRT-PCR involved the TaqMan probe-based real-time quantification system (Foster, CA, USA). The levels of mRNA were calculated relative to GAPDH mRNA as the invariant control. The sequences of primers are as following: human fetuin-A forward primer: 5′-CACAGGTAACAGCTCCGTGA-3′; human fetuin-A reverse primer: 5′-TGATTCCGCATACCCCAGTG-3′; human GAPDH forward primer: 5′-AACGGGAAGCTTGTCATCAATGGAAA-3′; human GAPDH reverse primer: 5′-GCATCAGCAGAGGGGGCAGAG-3′.
6
Quantifying ABCB11 gene expression
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Total RNA was isolated using NucleoZOL (Takara Cat. No. 740404.200) following manufacturer's instruction. cDNA was prepared from (deoxyribonuclease treated) total RNA using RevertAid Reverse Transcriptase (Thermo Cat. No. EP0441) following the manufacturer's instructions. Real Time PCR was done with unique oligonucleotide primers targeting
ABCB11 and
GAPDH, Ta=60°C, in triplicates and two repeats, using GoTaq® qPCR Master Mix (Promega Cat. No. A6001) following 'manufacturer's instructions on a Veriti Thermo Cycler from Applied Biosystems Waltham, Massachusetts, USA and data was acquired using the software associated with the same machine (ViiA7 V1.2) and relative quantification was calculated using the by 2
(–ΔΔCt) method. Oligonucleotide primer sequences are listed in
Table 3 .
An earlier version of this article can be found on biorxiv.org (DOI:
https://doi.org/10.1101/2020.09.01.277434 ).
ABCB11 and
GAPDH, Ta=60°C, in triplicates and two repeats, using GoTaq® qPCR Master Mix (Promega Cat. No. A6001) following 'manufacturer's instructions on a Veriti Thermo Cycler from Applied Biosystems Waltham, Massachusetts, USA and data was acquired using the software associated with the same machine (ViiA7 V1.2) and relative quantification was calculated using the by 2
(–ΔΔCt) method. Oligonucleotide primer sequences are listed in
An earlier version of this article can be found on biorxiv.org (DOI:
7
Optimized qPCR Protocol for Gene Expression Analysis
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Total RNA was isolated with NucleoZOL (Takara Bio USA, Mountain View, CA) according to the manufacturer's instructions. Reverse transcription reactions were done using hexamer and M-MuLV Reverse Transcriptase (New England Biolabs, Ipswich, MA). The cDNA products were diluted 20- to 100-fold and used as PCR templates. Primer3 Plus program were used to design the qPCR primer.57 (link) The qPCR analysis was carried out using our recently optimized TqPCR protocol.58 Briefly, the SYBR Green qPCR reactions (Bimake, Houston, TX) were set up according to manufacturer's instructions. The cycling program was modified by incorporating 4 cycles of touchdown steps prior to the regular cycling program as described58 : 95 °C × 3 min for one cycle; 95 °C × 20 s, 66 °C × 10 s for 4 cycles, with 3 °C decrease per cycle; followed by 95 °C × 10 s, 55 °C × 15 s, 70 °C × 1 s for 40 cycles, followed by plate read. All reactions were done in triplicate. GADPH was used as a reference gene. All sample values were normalized to GADPH expression by using the 2-ΔΔCt method. The qPCR primer sequences are listed in the Supplemental Table 1 .
8
Quantifying Chickpea ASR Gene Expression
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RNA isolation was done by using NucleoZOL (Takara Bio). Genomic DNA and other contaminants were removed by precipitation. One phase RNA extraction was followed by conversion to first strand cDNA using the Accuscript high fidelity cDNA synthesis kit (Agilent). Brilliant III ultra fast SYBR Green was used to measure the relative changes in the expression of chickpea ASR genes under water stressed conditions. The cDNA from leaf tissues were used as template. The Beta Actin gene was used as the reference gene. The qRT-PCR was conducted on a CFX 96 Real Time PCR (Biorad) in a reaction volume of 25μL, that comprised of 2μL chickpea samples cDNA, 0.5 μL each ASR specific forward and reverse primer, 12.5 μL Brilliant III ultra fast SYBR Green QPCR master mix (Agilent), and 9.5 μL nuclease-free molecular biology grade water. The qRT-PCR reaction cycle included 95°C for 3 min followed by 40 cycles at 95°C for 5 s, 60°C for 12s. The relative expression levels of chickpea ASR genes under different treatments were then calculated using the 2−ΔΔCT method [88 ].
9
RNA Extraction from Frozen Brain Tissues
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Mouse brain tissues were snap-frozen in liquid nitrogen immediately after dissection. Human brain tissues were snap-frozen in liquid nitrogen within an hour after dissection. All tissues were stored in liquid nitrogen thereafter. Total RNA was isolated from 20 mg frozen tissues, using NucleoZOL™ (Takara Bio, 740404.200) and NucleoSpin® RNA set for NucleoZOL™ (Takara Bio, 740406.50) following the manufactures specifications, followed by rDNase Set (Takara Bio, 740963) to digest DNA, and NucleoSpin® RNA Clean-up XS (Takara Bio, 740903) for RNA repurification. RNA purity (260/280, 260/230) and concentration were measured on NanoDrop™ 2000/2000c Spectrophotometers. RNA integrity number (RIN) was assessed using Agilent 2100 Bioanalyzer system.
10
Cell Sorting and RNA Isolation Protocol
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The method was developed based on a previously published study (46 (link)). A step-by-step protocol for sample preparation, sorting, and RNA isolation is provided in Text S1 . Key to successful RNA recovery is the gentle formaldehyde fixation at 4°C. Aliquots of fixed samples were adjusted to approximately 1.8 × 107 cells/mL in 30 mL volume each and stained with SYBR green. Sorting of 5.4 × 108 cells based on the FITC-signal (see above) directly into RNAprotect was performed with the BD FACSAria Fusion (BD Biosciences, Heidelberg, Germany). The sorted cells were collected on a filter from which RNA was extracted using a combination of Lysozyme and Proteinase K digestion with bead beating, and purified with NucleoZOL (TaKaRa Bio, Göteborg, Sweden). rRNA depletion was performed with the NEBNext Bacteria kit (NEB, Frankfurt, Germany). The libraries were prepared with the TruSeq kit (Illumina, San Diego USA).
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