Quick dna rna miniprep

Manufactured by Zymo Research

The Quick-DNA/RNA Miniprep is a lab equipment product from Zymo Research. It is a versatile tool designed for the rapid isolation and purification of both DNA and RNA from a variety of sample types. The product provides a simple and efficient method for extracting nucleic acids, allowing users to obtain high-quality samples for downstream applications.

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7 protocols using quick dna rna miniprep

Identifying Nuclear Expulsion Signature

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RNA was extracted from Padi4^KO cells co-cultured with NEPs or apoDBs, as well as Padi4^WT or Padi4^KO spheroids using Zymo Quick-DNA/RNA Miniprep. Samples were sequenced on an Illumina NextSeq. Quantification, quality control, differential expression and pathway analysis were performed using the CCBR Pipeliner (https://github.com/CCBR/Pipeliner).
To generate the nuclear expulsion signature, we identified 200 genes that were differentially expressed in both the co-culture and spheroid groups by selecting genes that were upregulated using log₂(FC) > 2 or downregulated using log₂(FC) < −2 and both with FDR < 0.0001. Among these 200 genes, 28 genes were selected as nuclear expulsion signature by gene function, upstream or downstream of nuclear expulsion and single gene clinical relevance. Finally, the 28-gene signature was examined for clinical correlation using significance for DMFS in KMPLOT, GSE2603 +2034 of breast cancer as well as OS in CaArray and GSE37745 of lung cancer cohorts. A Cox proportional hazards model was used to validate significance and specificity of nuclear expulsion signature.

Park W.Y., Gray J.M., Holewinski R.J., Andresson T., So J.Y., Carmona-Rivera C., Hollander M.C., Yang H.H., Lee M., Kaplan M.J., Cappell S.D, & Yang L. (2023). Apoptosis-induced nuclear expulsion in tumor cells drives S100a4-mediated metastatic outgrowth through the RAGE pathway. Nature Cancer, 4(3), 419-435.

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Erythroid Differentiation and Transcriptome Analysis

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Total RNA was extracted from SCD or β-thalassemic HSPCs differentiated towards the erythroid lineage (day 13) or from healthy donor (HD) HSPCs (12 and 24 h post-transfection) using RNeasy micro kit (QIAGEN), and from BFU-E pools and single colonies using Quick-DNA/RNA Miniprep (ZYMO Research). RNA was treated with DNase using the DNase I kit (Invitrogen), following the manufacturer’s instructions. Mature transcripts were reverse-transcribed using SuperScript First-Strand Synthesis System for RT-qPCR (Invitrogen) with oligo (dT) primers. RT-qPCR was performed using the iTaq universal SYBR Green master mix (Bio-rad) and the Viia7 Real-Time PCR system (ThermoFisher Scientific), or the CFX384 Touch Real-Time PCR Detection System (Bio-rad). Supplementary Table 6 lists the primers used for RT-qPCR.

Antoniou P., Hardouin G., Martinucci P., Frati G., Felix T., Chalumeau A., Fontana L., Martin J., Masson C., Brusson M., Maule G., Rosello M., Giovannangeli C., Abramowski V., de Villartay J.P., Concordet J.P., Del Bene F., El Nemer W., Amendola M., Cavazzana M., Cereseto A., Romano O, & Miccio A. (2022). Base-editing-mediated dissection of a γ-globin cis-regulatory element for the therapeutic reactivation of fetal hemoglobin expression. Nature Communications, 13, 6618.

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Isolation and cDNA Synthesis from Neuronal RNA

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Neuronal lysates that were stored in DNA/RNA shield (Zymo Research) were thawed at RT and resuspended in an equal volume of lysis buffer (Zymo Research) just before starting the Quick-DNA/RNA miniprep (Zymo Research) protocol following the manufacturer instructions. Trace gDNA was removed from RNA samples using Monarch^® gDNA removal columns (Bioké), and this was followed by a treatment with TURBO^TM DNase (Thermo Fisher Scientific, Waltham, MA, USA). Purity of gDNA and RNA samples was checked using a NanoDrop^TM 2000 spectrophotometer (Thermo Scientific). RNA samples were immediately used for preparation of cDNA using the SuperScript^TM IV First-Strand Synthesis System (Invitrogen^TM, 18091200) with oligo(dT)₂₀ primers (Invitrogen^TM).

Boeren M., Van Breedam E., Buyle-Huybrecht T., Lebrun M., Meysman P., Sadzot-Delvaux C., Van Tendeloo V.F., Mortier G., Laukens K., Ogunjimi B., Ponsaerts P, & Delputte P. (2022). Activation of Interferon-Stimulated Genes following Varicella-Zoster Virus Infection in a Human iPSC-Derived Neuronal In Vitro Model Depends on Exogenous Interferon-α. Viruses, 14(11), 2517.

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Quantification of Latent Viral Infection

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Infectious centers were quantitated in CD34⁺ HPCs, as described previously (39). Frequency of infection centers were calculated using extreme limiting dilution analysis (42). For the investigation of USP1 activity during latency establishment, CD34⁺ HPCs were treated with 1 μM C527 (ApexBio) after sorting for CD34⁺ GFP⁺ populations and when stromals were replaced at 6 dpi. The study for USP1’s role in reactivation, 1 μM C527 was added at the time of reactivation. Proliferation of CD34⁺ cells during chemical inhibition was calculated by observing the fold change in the number of cells prior to and after inhibition for each condition. To understand JAK1/2’s role in latency establishment, CD34+ cells were treated with 500 nM Ruxolitinib (STEMCELL Technologies) every 24 hours for 5 days. Cells were lysed and DNA was extracted at days 1 and 5 utilizing the Quick-DNA/RNA Miniprep (Zymo Research). Viral genomes were quantified using a standard curve and normalized to RNAseP.

Zarrella K., Longmire P., Zeltzer S., Collins-McMillen D., Hancock M., Buehler J., Reitsma J.M., Terhune S.S., Nelson J.A, & Goodrum F. (2023). Human cytomegalovirus UL138 interaction with USP1 activates STAT1 in infection. PLOS Pathogens, 19(6), e1011185.

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Genome Editing Analysis in Erythroid Cells

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Genome editing was analyzed in HUDEP-2 cells at days 0 and 9 of erythroid differentiation and in CB and adult mobilized HSPC-derived erythroid cells at days 6 and 14 of erythroid differentiation, respectively. Genomic DNA was extracted from control and edited cells using the PureLink Genomic DNA Mini Kit (Life Technologies), Quick-DNA/RNA Miniprep (ZYMO Research), or DNA Extract All Reagents Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. To evaluate NHEJ efficiency at gRNA target sites, we performed PCR followed by Sanger sequencing and TIDE analysis (tracking of InDels by decomposition) (49 (link)) or ICE CRISPR Analysis Tool (Synthego) (Table 2) (50 (link)).
Digital droplet PCR was performed using EvaGreen mix (Bio-Rad) to quantify the frequency of the 4.9-kb deletion. Short (~1 min) elongation time allowed the PCR amplification of the genomic region harboring the deletion. Control primers annealing to a genomic region on the same chromosome (chr 11) were used as DNA loading control (Table 3).

Weber L., Frati G., Felix T., Hardouin G., Casini A., Wollenschlaeger C., Meneghini V., Masson C., De Cian A., Chalumeau A., Mavilio F., Amendola M., Andre-Schmutz I., Cereseto A., El Nemer W., Concordet J.P., Giovannangeli C., Cavazzana M, & Miccio A. (2020). Editing a γ-globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype. Science Advances, 6(7), eaay9392.

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Quantifying Viral Latency and Reactivation Mechanisms

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Infectious centers were quantitated in CD34⁺ HPCs, as described previously (39). Frequency of infection centers were calculated using extreme limiting dilution analysis (42). For the investigation of USP1 activity during latency establishment, CD34⁺ HPCs were treated with 1uM/mL C527 (ApexBio) after sorting for CD34⁺ GFP⁺ populations and when stromals were replaced at 6 dpi. The study for USP1’s role in reactivation, 1uM/mL C527 was added at the time of reactivation. Proliferation of CD34⁺ cells during chemical inhibition was calculated by observing the fold change in the number of cells prior to and after inhibition for each condition. To understand JAK1/2’s role in latency establishment, CD34+ cells were treated with 500nM Ruxolitinib (STEMCELL Technologies) every 24 hours for 5 days. Cells were lysed and DNA was extracted at days 1 and 5 utilizing the Quick-DNA/RNA Miniprep (Zymo Research). Viral genomes were quantified using a standard curve and normalized to RNAseP.

Zarrella K., Longmire P., Zeltzer S., Collins-McMillen D., Hancock M., Buehler J., Reitsma J.M., Terhune S.S., Nelson J.A, & Goodrum F. (2023). Human Cytomegalovirus UL138 Interaction with USP1 Activates STAT1 in infection. bioRxiv.

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RNA Extraction and cDNA Synthesis from RNA-Shield Samples

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RNA extraction and cDNA synthesis of samples stored in DNA/RNA shield were performed as described previously (9 (link)). In brief, samples were thawed at RT and resuspended in an equal volume of lysis buffer (Zymo Research) before starting the Quick-DNA/RNA miniprep (Zymo Research) protocol following the manufacturer’s instructions. After a TURBO™ DNase (Thermo Fisher Scientific) treatment step, purity of RNA samples was checked using a NanoDrop™ 2000 spectrophotometer (Thermo Scientific) and cDNA synthesis was carried out using the SuperScript™ IV First-Strand Synthesis System (Invitrogen™) with oligo(dT)₂₀ primers (Invitrogen™).

Van Breedam E., Buyle-Huybrecht T., Govaerts J., Meysman P., Bours A., Boeren M., Di Stefano J., Caers T., De Reu H., Dirkx L., Schippers J., Bartholomeus E., Lebrun M., Sadzot-Delvaux C., Rybakowska P., Alarcón-Riquelme M.E., Marañón C., Laukens K., Delputte P., Ogunjimi B, & Ponsaerts P. (2023). Lack of strong innate immune reactivity renders macrophages alone unable to control productive Varicella-Zoster Virus infection in an isogenic human iPSC-derived neuronal co-culture model. Frontiers in Immunology, 14, 1177245.

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