Direct zol rna miniprep kit

Manufactured by Zymo Research

Sourced in United States, Germany, Switzerland, United Kingdom

The Direct-zol RNA MiniPrep kit is a product offered by Zymo Research for the extraction and purification of RNA from various sample types. It utilizes a rapid spin-column-based method to isolate high-quality RNA.

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2 158 protocols using direct zol rna miniprep kit

Isolation and Detection of circRNAs

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Total RNA and RNA from nuclear/cytoplasmic fractionations was isolated using the Direct-zol RNA MiniPrep Kit with on-column DNAse treatment, according to the manufacturer's instructions (Zymo Research).
RNaseR treatment was performed on total RNA extracted from mouse EBs at day 6 of differentiation; 6 U of RNaseR (RNR07250, Epicentre) was used for 1 μg of RNA and the reaction was carried out for 15′ at 37 °C; the RNA was then extracted using the Direct-zol RNA MiniPrep Kit (Zymo Research).
For RNA retro-transcription, the SuperScript VILO cDNA Synthesis Kit was used (Thermo Fisher Scientific).
For the detection of circRNAs and their linear counterparts, cDNA samples were analysed by quantitative real-time PCR using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific).
For the RNA extracted in CLIP experiments, semi-quantitative PCR was performed on cDNAs using MyTaq Red DNA Polymerase (Bioline) according to the manufacturer's instructions. The samples were then loaded on a 2.5% agarose gel. All the images were captured using the Molecular Imager ChemiDoc XRS+ (Bio-Rad), and the densitometric analyses were performed using the associated Image Lab software (Bio-Rad).
The oligonucleotides used in all the amplification steps are listed in Supplementary Table 1.

Errichelli L., Dini Modigliani S., Laneve P., Colantoni A., Legnini I., Capauto D., Rosa A., De Santis R., Scarfò R., Peruzzi G., Lu L., Caffarelli E., Shneider N.A., Morlando M, & Bozzoni I. (2017). FUS affects circular RNA expression in murine embryonic stem cell-derived motor neurons. Nature Communications, 8, 14741.

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Quantitative PCR Analysis of Skin Cells

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RNA was purified from FACS sorted cells by directly sorting into TrizolLS (Invitrogen) and purified using Direct-zol RNA MiniPrep kit (Zymo Research). Equivalent amounts of RNA were reverse-transcribed by SuperScript VILO cDNA Synthesis Kit (Invitrogen). cDNAs were normalized to equal amounts using primers against β-actin. cDNAs were mixed with indicated primers and Power SYBR Green PCR Master Mix (Applied Biosystems), and quantitative PCR (qPCR) was performed on a Applied Biosystems 7900HT Fast Real-Time PCR system. Primer sequences for RT-PCR were obtained from Roche Universal ProbeLibrary.
For Vγ5 qPCR, unwounded and wounded skin was incubated in 50mM EDTA for 1hour, to separate epidermis was separated from dermis. Epidermal cells were immediately frozen in liquid nitrogen. Frozen tissues were homogenized using Bessman Tissue Pulverizer (SpectrumTM) and collected in Trizol (Invitrogen). RNA was extracted using Direct-zol RNA MiniPrep kit (Zymo Research) per manufacturer’s instructions. Equivalent amounts of RNA were reverse-transcribed by SuperScript VILO cDNA Synthesis Kit (Invitrogen). cDNAs were mixed with indicated primers and Power SYBR Green PCR Master Mix (AppliedBiosystems), and quantitative PCR (qPCR) was performed on a Applied Biosystems 7900HT Fast Real-Time PCR system.

Keyes B.E., Liu S., Asare A., Naik S., Levorse J., Polak L., Lu C.P., Nikolova M., Pasoli H.A, & Fuchs E. (2016). Impaired Epidermal to Dendritic T-Cell Signaling Slows Wound Repair in Aged Skin. Cell, 167(5), 1323-1338.e14.

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Comprehensive RNA Extraction from Plant Tissues

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For each plant species, four infected samples and one mock inoculated sample were randomly selected out of ten replicates for total RNA extraction and downstream analysis using the different quantification techniques. For N. tabacum, RNA was extracted from 100 μl of homogenised leaf tissue using the RNeasy Plant Mini Kit (Qiagen), following the manufacturer's protocol (with on-column DNAse digestion). For C. quinoa, RNA was extracted from 200 μl of homogenised leaf tissue using the Direct-zol RNA miniprep kit (Zymo), following the manufacturer's protocol. For N. benthamiana, RNA was extracted from 100 μl or 200 μl of homogenised leaf tissue using the Direct-zol RNA miniprep kit (Zymo), following the manufacturer's protocol. For all samples, RNA was eluted in 40 μl RNase free water, and yield and purity were determined using the Nanodrop spectrophotometer and Qubit3 fluorometer (RNA HS Assay kit, Invitrogen). After extraction, multiple aliquots of RNA were stored at −80 °C.

Boezen D., Johnson M.L., Grum-Grzhimaylo A.A., van der Vlugt R.A, & Zwart M.P. (2023). Evaluation of sequencing and PCR-based methods for the quantification of the viral genome formula. Virus Research, 326, 199064.

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Isolation of Total RNA from Serum Samples

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Total RNA in serum samples was isolated using the Direct-zol RNA MiniPrep Kit according to the procedures specified in the manufacturer’s protocol (Zymo Research, Irvine, CA, USA). An equal volume of room temperature 2 × denaturing Trizol reagent was added to 700 µL of a 32 Units-volume sample and immediately vortexed. The vortexed samples were then centrifuged at 12,000 rpm for 10 min at 4 °C to separate the organic phase. The upper clear phase was transferred to a new tube. Next, room temperature 100% ethanol, at a volume 1.25 times the sample volume, was added to the tube and mixed well. This mixture (ethanol/lysate) was then filtered. The filter was washed with 700 µL of miRNA Wash 1 Solution. This procedure was repeated twice with 2/3 solution. Finally, RNA was collected from the filter in 50 µL RNAase/DNAase-free water. Concentration and quality assessments of the obtained RNA were performed using a spectrophotometer (Thermo Scientific Multiskan GO, Vantaa, Finland). The samples were considered adequate if their A260/A280 ratio was in the range between 1.8 and 2.0. The RNA isolation kit (Direct-zol RNA MiniPrep Kit, Zymo Research) was used in compliance with the manufacturer’s instructions for isolating total RNA from isolated and stored cardiomyocytes.

Tas D., Kaplan O, & Sogut O. (2020). Validity of Serum miRNA 93 and miRNA 191 to Reduce Unnecessary Computed Tomography in Patients With Mild Head Trauma. Journal of Clinical Medicine Research, 12(9), 579-589.

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Parallel RNA-seq and Ribo-seq Analysis of Combination Therapy

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For RNA-seq performed in parallel with Ribo-seq, RNA was isolated by Direct-zol RNA MiniPrep Kit (Zymo Research) from 100 µL of cell lysates after homogenization by syringe and clearing by centrifugation. For combination WINi/BETi treatment, MV4;11 cells were treated for 48 hr with either 0.2% DMSO, 100 nM C16, 2.5 nM mivebresib, or combined 100 nM C16 and 2.5 nM mivebresib, and RNA isolated by Direct-zol RNA MiniPrep Kit (Zymo Research) with on-column DNAse-treatment. For RNA-seq in MV4;11 NT and RPL22 KO cells, cultures were treated for 48 hr with either 0.1% DMSO or 100 nM C16 before RNA isolation as described above for WINi/BETi RNA-seq. For all RNA-seq experiments, RNA was submitted to the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core facility for library preparation with rRNA-depletion using standard Illumina protocols and sequencing on an Illumina NovaSeq 6000.

Howard G.C., Wang J., Rose K.L., Jones C., Patel P., Tsui T., Florian A.C., Vlach L., Lorey S.L., Grieb B.C., Smith B.N., Slota M.J., Reynolds E.M., Goswami S., Savona M.R., Mason F.M., Lee T., Fesik S., Liu Q, & Tansey W.P. (2024). Ribosome subunit attrition and activation of the p53–MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition. eLife, 12, RP90683.

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RNA Extraction and qRT-PCR Analysis

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Lungs were homogenized in TRI Reagent with 1.0 mm diameter zirconia beads (Biospec Products) for 30 seconds using bead beater disruption (Minibeadbeater-16, BioSpec Products). RNA was extracted as described in the manufacturer’s protocol (Direct-zol RNA miniprep kit, Zymo Research). First-strand complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) at 25°C for 10 min, 37°C for 120 min, 85°C for 5 min, followed by a cooling step at 4°C. qRT-PCR was performed using SYBR Green Master Mix (Applied Biosystems) with the following thermocycling program: [95°C for 5 min, 95°C for 15 sec, 55°C for 1 min] x 40 times, followed by 65°C to 95°C (0.5°C increment) for 5 sec (C1000 Touch Thermal Cycler, Bio Rad). Hprt1 was used as the housekeeping gene. During RNA isolate, RNA was treated with DNase using the Direct-zol RNA miniprep kit from Zymo Research. RNA (without reverse transcriptase treatment) was tested as a genomic contamination control. 1 ug of total RNA was used for the first strand cDNA template synthesis to generate 20 μl of cDNA. cDNA was dilution by a factor of 4 and 1 μl was used in a 10 μl RT-PCR reaction. Primer sequences are provided in S1 Table.

Park S.S., Mai M., Ploszaj M., Cai H., McGarvey L., Mueller C., Garcia-Arcos I, & Geraghty P. (2023). Type 1 diabetes contributes to combined pulmonary fibrosis and emphysema in male alpha 1 antitrypsin deficient mice. PLOS ONE, 18(10), e0291948.

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Parallel RNA-Seq and Ribo-Seq Protocols

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For RNA-Seq performed in parallel with Ribo-Seq, RNA was isolated by Direct-zol RNA MiniPrep Kit (Zymo Research) from 100 μL of cell lysates after homogenization by syringe and clearing by centrifugation. For combination WINi/BETi treatment, MV4;11 cells were treated for 48 hours with either 0.2% DMSO, 100 nM C16, 2.5 nM mivebresib, or combined 100 nM C16 and 2.5 nM mivebresib, and RNA isolated by Direct-zol RNA MiniPrep Kit (Zymo Research) with on-column DNAse-treatment. For RNA-Seq in MV4;11 NT and RPL22 KO cells, cultures were treated for 48 hours with either 0.1% DMSO or 100 nM C16 before RNA isolation as described above for WINi/BETi RNA-Seq. For all RNA-Seq experiments, RNA was submitted to the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core facility for library preparation with rRNA-depletion using standard Illumina protocols and sequencing on an Illumina NovaSeq 6000.

Howard G.C., Wang J., Rose K.L., Patel P., Tsui T., Florian A.C., Lorey S.L., Grieb B.C., Smith B.N., Slota M.J., Reynolds E.M., Goswami S., Savona M.R., Lee T., Fesik S.W., Liu Q, & Tansey W.P. (2023). Ribosome subunit attrition and activation of the p53–MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition. bioRxiv.

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RNA Extraction and Purification from AAV-Infected Cells

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Cell debris in the lysate was pelleted by centrifugation at 4,000 rpm for 5 minutes. The supernatant was transferred into a new 15 ml tube and the total RNA was isolated using the Direct-zol RNA MiniPrep kit (Zymo) according to the manufacturer’s instructions with the following modifications. The lysate from 10 million cells was split onto 2 RNA purification spin-columns in order to not exceed the RNA binding capacity of a column. Elution was done in 50 μl RNase-free H2O per column and the two eluates from 10 million cells were pooled afterwards.
In order to digest traces of co-isolated AAV genomes, total RNA was treated with TURBO DNase (Invitrogen) for 30 minutes at 37 °C and subsequently cleaned-up using the Direct-zol RNA MiniPrep kit (Zymo) for purification. One column was used per sample. Elution was in 25 μl RNase-free H₂O.

Herholt A., Brankatschk B., Kannaiyan N., Papiol S., Wichert S.P., Wehr M.C, & Rossner M.J. (2018). Pathway sensor-based functional genomics screening identifies modulators of neuronal activity. Scientific Reports, 8, 17597.

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Total RNA Isolation and RT-PCR Analysis

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Total RNA was isolated from cultured cells and mice tissues. For tissues, hearts from WT and mutant mice were mechanistically reduced to a pulp and resuspended in 500 μL of TRI Reagent (Zymo Research). Samples were centrifuged for 30 min at 16,000 x g at 4°C to collect the supernatant. RNA was isolated using Direct-zol RNA MiniPrep Kit (Zymo Research) according to manufacturer’s instructions and quantified by Nanodrop (Thermo Scientific). From C₂C₁₂ cells, total RNA was isolated using TRI Reagent (Zymo Research) followed by column purification using Direct-zol RNA MiniPrep Kit (Zymo Research) and quantified by Nanodrop (Thermo Scientific). For semiquantitative and quantitative RT-PCR analyses, RNA (0.5-1.0 μg) was reverse transcribed using PrimeScript Reagent Kit (Takara), according to manufacturer’s instructions. Amplification by PCR was carried out using Mytaq (Bioline) (RT-PCR) or PowerUp SYBR-Green MasterMix (Thermo Fisher Scientific) (RT-qPCR) reagents. See Table S1 for oligo and Key Resources Table for reagents details.

Desideri F., Cipriano A., Petrezselyova S., Buonaiuto G., Santini T., Kasparek P., Prochazka J., Janson G., Paiardini A., Calicchio A., Colantoni A., Sedlacek R., Bozzoni I, & Ballarino M. (2020). Intronic Determinants Coordinate Charme lncRNA Nuclear Activity through the Interaction with MATR3 and PTBP1. Cell Reports, 33(12), 108548.

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Bacterial RNA Extraction and Purification

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Total RNA was extracted from 10 to 20 ml of bacterial culture by washing the cells in guanidinium thiocyanate buffer, bead beating washed pellets into TRIzol, extracting RNA in chloroform, and purifying extracted RNA with the Direct-zol RNA miniprep kit (Zymo Research, R2052). After elution, DNA was removed from total RNA samples by incubation with TURBO DNase (Ambion, AM2238) at 37°C for 1 hour. Total RNA samples were then repurified using the RNA Clean and Concentrator-25 kit (Zymo Research, R1017) for RNA sequencing (RNA-seq) samples or the Direct-zol RNA miniprep kit for qRT-PCR samples.

Schrader S.M., Botella H., Jansen R., Ehrt S., Rhee K., Nathan C, & Vaubourgeix J. (2021). Multiform antimicrobial resistance from a metabolic mutation. Science Advances, 7(35), eabh2037.

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