Direct zol miniprep kit

Manufactured by Zymo Research

Sourced in United States

The Direct-zol miniprep kit is a tool designed for the rapid and efficient extraction and purification of high-quality total RNA from a variety of sample types, including cells, tissue, and microorganisms. The kit utilizes a direct-to-Trizol method, eliminating the need for sample handling and minimizing RNA loss.

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88 protocols using direct zol miniprep kit

RNA Amplification and Purification

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TUG1 amplicon was reamplified using a forward primer with a T7 sequence. After purification, 100 ng of DNA were used for in vitro transcription using T7 polymerase and rNTPs from Takara (Clonthech) following manufacturer’s instructions. The IVT product was purified using the Direct-zol miniprep kit (Zymo Research).

Castellanos-Rubio A., Santin I., Olazagoitia-Garmendia A., Romero-Garmendia I., Jauregi-Miguel A., Legarda M, & Bilbao J.R. (2019). A novel RT-QPCR-based assay for the relative quantification of residue specific m6A RNA methylation. Scientific Reports, 9, 4220.

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Quantifying XIST and TAF1 Expression in iPSCs

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Total RNA was extracted from iPSCs with a Direct-zol Miniprep kit (Zymo Research). For reverse transcription, we used SuperScript IV VILO Master Mix (Thermo Fisher Scientific). The expression of XIST and TAF1 was analyzed via qPCR with a QuantiTect SYBR Green PCR kit (Qiagen) on a QuantStudio 3 (Applied Biosystems). The following 5 primer sets were used: XIST Forward: 5'-TTGCCCTACTAGCTCCTCGGAC-3'; XIST Reverse: 5'-TTCTCCAGATAGCTGGCAACC-3';

D’Ignazio L., Jacomini R.S., Qamar B., Benjamin K.J., Arora R., Sawada T., Evans T.A., Diffenderfer K.E., Pankonin A.R., Hendriks W.T., Hyde T.M., Kleinman J.E., Weinberger D.R., Bragg D.C., Paquola A.C., & Erwin J.A. (2022). Variation in TAF1 expression in female carrier induced pluripotent stem cells and human brain ontogeny has implications for adult neostriatum vulnerability in X-linked Dystonia Parkinsonism.

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HIV Entry and RNA Extraction Protocol

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TZM-b1 or Jurkat cells were treated with nevirapine at 1 μM/ml for 2 h incubated at 37°C and with HIV-1 (wild type or mutant) for 2 h at 4°C to facilitate virus attachment and then at 37°C for 2 h to initiate fusion and entry. Cellular nucleic acids were collected using Trizol reagent (Life Technologies) and the RNA fraction was column purified using a Direct-zol mini-prep kit (Zymo Research) and treated on the column with DNase I prior to elution. The purified RNAs were used in RT-PCR reactions both with and without SuperScript III reverse transcriptase (Life Technologies) using random hexamer oligonucleotides for the first strand DNA synthesis. Second strand DNA and PCR was performed using oligonucleotides specific for full length viral mRNA as previously described [4 (link)]. A no template control was included in each experiment.

Li D., Wei T., Rawle D.J., Qin F., Wang R., Soares D.C., Jin H., Sivakumaran H., Lin M.H., Spann K., Abbott C.M, & Harrich D. (2015). Specific Interaction between eEF1A and HIV RT Is Critical for HIV-1 Reverse Transcription and a Potential Anti-HIV Target. PLoS Pathogens, 11(12), e1005289.

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Gene Expression Analysis in Mice Livers

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A small portion of liver from each mouse was cut and placed in RNAlater (Invitrogen). Total RNA was isolated using TRIzol Reagent (Life Technologies) and Direct-Zol MiniPrep Kit (Zymo). cDNA was generated from 500ng of RNA using iScript™ Reverse Transcription Supermix for RT-qPCR (Bio-Rad). qPCR was performed on a total of 5.6ng of cDNA per sample using Power SYBR™ Green PCR Master Mix (Applied Biosystems) on the CFX384 Real-Time System (Bio-Rad). Fold changes in gene expression were calculated using the Ct method from Ct values normalized to Gapdh on Microsoft Excel and PRISM. Primer sequences for each gene were published as follows:
Mouse GS F - 5′-ATGCAGATAGGGTGACCACT-3′
Mouse GS R - 5′-GTCCATTTGCAGGAAATGGC-3′
Mouse Axin2 F - 5′-GCAGGAGCCTCACCCTTC-3′
Mouse Axin2 R - 5′-TGCCAGTTTCTTTGGCTCTT-3′
Mouse Gapdh F - 5′-CCCCAATGTGTCCGTCGTG-3’
Mouse Gapdh R - 5′-GCCTGCTTCACCACCTTCT-3′

Dang L.T., Miao Y., Ha A., Yuki K., Park K., Janda C.Y., Jude K.M., Mohan K., Ha N., Vallon M., Yuan J., Vilches-Moure J.G., Kuo C.J., Garcia K.C, & Baker D. (2019). Receptor subtype discrimination using extensive shape complementary designed interfaces. Nature structural & molecular biology, 26(6), 407-414.

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Maternally Deposited sisRNA and mRNA Extraction

Cited 3 times

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RNA extraction was performed as described previously [15 (link)], using TRIzol (Ambion) and the Direct-zol miniprep kit (Zymo Research). To detect maternally deposited sisRNA and mRNA, stage 14 oocytes were manually isolated to avoid contamination with pre-mRNA in transcriptionally active germline cells. For experiments involving detection of pre-mRNA and mRNA for gene expression and splicing efficiency, whole ovaries were used as they reflect the steady-state levels of pre-mRNA and mRNA as described in previous studies [32 (link),33 (link)].

Voo K., Ching J.W., Lim J.W., Chan S.N., Ng A.Y., Heng J.Y., Lim S.S, & Pek J.W. (2021). Maternal starvation primes progeny response to nutritional stress. PLoS Genetics, 17(11), e1009932.

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Gene Expression Profiling by qRT-PCR

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Cells were harvested with Trizol reagent and stored at -80 °C until use. Total RNA was extracted using the Direct-zol Miniprep kit (Zymo Research, R2052). cDNA was prepared using the M-MLV reverse transcriptase (Invitrogen, 28025-013) per manufacturer's instruction. Quantitative PCR was performed on ViiA 7 Real-time PCR system (Applied Biosystem) using SYBR-Green Master PCR Mix (Clontech, 639676) in triplicates. All quantifications were normalized to an endogenous GAPDH control (for primer sequences, please refer to Supplementary Table 3).

Shao Z., Zhang R., Khodadadi-Jamayran A., Chen B., Crowley M.R., Festok M.A., Crossman D.K., Townes T.M, & Hu K. (2016). The acetyllysine reader BRD3R promotes human nuclear reprogramming and regulates mitosis. Nature Communications, 7, 10869.

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Differential Gene Expression Analysis of KSHV-Infected Cells

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Total RNA was extracted using Direct-Zol Miniprep kit (Zymo), following manufacturer’s instructions. Library preparation and sequencing was performed by NCI CCR Sequencing facility. Ribosomal RNA was removed, and sequencing libraries were prepared using Illumina TruSeq Stranded / NEBnext Ultra Low Input Total RNA Library Prep and paired-end sequencing. The reads from the FASTQ files (reads were generated by HiSeq4000) were trimmed of Illumina adapters using the Fastx toolkit. The reads were aligned to two target genomes, human (GRCh38) and KSHV (Genbank accession number NC_009333.1) using TopHat^{49 (link)}. Transcripts were assembled using Cufflinks and Cuffdiff^{50 (link)} in order to reveal differentially expressed genes. Significant mRNA fold change was determined by an adjusted p-value ≤ 0.05 based on the Benjamini and Hochberg multiple testing correction. DEGs from JSC-1 group and BCBL-1 group were determined by fold change ≥ 2, adjusted p-value < 0.05. Pathway enrichment analysis was performed with Ingenuity Pathway Analysis (IPA, Qiagen). Enriched pathways with p < 0.05 and |Z-score|≥ 1 were selected and presented using GraphPad Prism 9. GraphPad Prism 9 was also used for visualization of heat maps and scatter plots.

Wu Y., Wang V, & Yarchoan R. (2024). Pacritinib inhibits proliferation of primary effusion lymphoma cells and production of viral interleukin-6 induced cytokines. Scientific Reports, 14, 4125.

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HCT116 Cell Total RNA Extraction

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To lyse and isolate the total RNA of HCT116 cell lines, TRIzol^TM Reagent (Life Technologies, Carlsbad, CA, USA) and the Direct-zol^® MiniPrep kit (Zymo Research, Irvine, CA, USA) were used. DNA contamination is eliminated following a 15 min DNAse I (Zymo Research, Irvine, CA, USA) treatment. The quality and quantity of the RNAs were determined using the nanophotometer IMPLEN (IMPLEN, München, Germany). Complementary DNA (cDNA) was synthesized from 1 µg of total RNA using the First Strand cDNA Synthesis Kit for RT-PCR (AMV) (Roche, Basel, Switzerland) and SuperScript™ III Reverse Transcriptase (Life Technologies, Paisley, UK). The real-time PCR experiments were carried out using TaqMan™ Fast Advanced Master Mix (Life Technologies, Paisley, UK) by QuantStudio™ 5 and 6 Pro Real-Time PCR System (Life Technologies, Paisley, UK). To determine the relative expression, three replicates were used based on a 2^−ΔΔCt method for fold change determination [39 (link)]. Probes of TaqMan^® assays provided by Thermo Fisher Scientific are listed in Supplementary Table S4B.

Jacksi M., Schad E, & Tantos A. (2024). Morphological Changes Induced by TKS4 Deficiency Can Be Reversed by EZH2 Inhibition in Colorectal Carcinoma Cells. Biomolecules, 14(4), 445.

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Quantifying cif Gene Expression in Aging Flies

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To test the effects of male age on cif gene expression, siblings of males used in hatch rates were collected and frozen at −80°C. RNA was extracted from individual, whole flies using Direct-zol MiniPrep kit (Zymo Research) and treated with DNA-Free DNase Treatment and Removal Kit (Invitrogen). Steps involving Trizol were conducted in a chemical fume hood. RNA was then converted to cDNA using the Superscript VILO cDNA Synthesis Kit (Invitrogen) and RT-qPCR was conducted using iTaq Universal SYBR Green Supermix (Bio-Rad). Primers were used as listed in previous literature (LePage et al., 2017 (link); Shropshire et al., 2018 (link)), and the conditions were as follows: 95°C 5 min, 40x (95°C 10s, 59°C 1 min), melt curve from 35°C to 95°C, increment 0.5°C. Variation in gene expression across ages was quantified using 2^-Δct (the difference in ct values between rp49 and cif gene expression levels).

Ritchie I.T., Needles K.T., Leigh B.A., Kaur R, & Bordenstein S.R. (2022). Transgenic cytoplasmic incompatibility persists across age and temperature variation in Drosophila melanogaster. iScience, 25(11), 105327.

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RNA Affinity Purification and Sequencing

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RNA Affinity purification was performed using 1 g of cell powder per triplicate as described above but without crosslinking. Lysates were incubated with either IgG- or GFP-nanobody-conjugated magnetic beads for 30 min. Beads were washed extensively (8 times) in extraction buffer before being resuspended in 1 ml of Trizol (Invitrogen, 15596026) and vortexed vigorously. Control experiments were carried out using strains expressing Protein A or GFP alone, and RNAs identified in these samples were considered background and filtered from those identified with Mlp1-PrA and Nup133-GFP, respectively. The total poly(A) library was generated by RNA extraction using 100 mg of cryo-ground cell powder thawed into Trizol in triplicates. RNA was then extracted using the Direct-zol Miniprep Kit (Zymo Research, R2050) and Dnase treatment was performed on-column according to the manufacturer’s instructions. Samples were resuspended in 30 μl ultra-pure water (Invitrogen, 10977023), and the quality of RNA was assessed by Qbit and Bioanalyzer chip. RNA extracts were Poly(A)-RNA enriched via a Poly(A) mRNA magnetic Isolation Module oligo-dT (NEBNext), and cDNA libraries were prepared using the Kapa RNA HyperPrep Kit (96 rxns, Roche) and TruSeq DNA UDI 96 indexes (Illumina). RNA-sequencing was performed using Nocaseq6000 flowcell S2 PE50.

Bensidoun P., Reiter T., Montpetit B., Zenklusen D, & Oeffinger M. (2022). Nuclear mRNA metabolism drives selective basket assembly on a subset of nuclear pore complexes in budding yeast. Molecular cell, 82(20), 3856-3871.e6.

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