Allprep dna rna mini kit

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The AllPrep DNA/RNA Mini Kit is a laboratory product designed to simultaneously purify high-quality genomic DNA and total RNA from a single biological sample. The kit utilizes a spin-column-based method to ensure efficient extraction and separation of both DNA and RNA components.

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1 681 protocols using allprep dna rna mini kit

Isolation and Quantification of Nucleic Acids

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DNA of tissue and organoids was isolated using the AllPrep DNA/RNA Mini Kit (QIAGEN) according to manufacturers’ protocol. DNA purifications were quantified by measuring optical density at 260‐nm wavelengths.
RNA from cells of 3D cultures was isolated using the RNeasy Mini Kit (QIAGEN) or the AllPrep DNA/RNA Mini Kit (QIAGEN). RNA from tissue samples was isolated using the AllPrep DNA/RNA Mini Kit (QIAGEN). After pelleting the organoids, they were lysed in the respective sample buffer and RNA purification was performed according to the manufacturer's protocols. RNA concentration and purity were measured with a NanoDrop® ND‐1000 Spectrophotometer.
For microarray analysis, total RNA was isolated with TRIzol (Life Technologies) according to the supplier's protocol using glycogen as carrier. Quality control and quantification of total RNA were carried out using the NanoDrop 1000 UV‐Vis spectrophotometer (Kisker) as well as the Agilent 2100 Bioanalyzer with a RNA Nano 6000 microfluidics kit (Agilent Technologies).

Hoffmann K., Berger H., Kulbe H., Thillainadarasan S., Mollenkopf H., Zemojtel T., Taube E., Darb‐Esfahani S., Mangler M., Sehouli J., Chekerov R., Braicu E.I., Meyer T.F, & Kessler M. (2020). Stable expansion of high‐grade serous ovarian cancer organoids requires a low‐Wnt environment. The EMBO Journal, 39(6), e104013.

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Attenuated and Virulent N. perurans DNA Extraction

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DNA was extracted using the AllPrep DNA/RNA Mini Kit (QIAGEN, Hilden, Germany) from the attenuated and virulent N. perurans cultures in triplicate. Three plates of the attenuated and virulent cultures were individually prepped for DNA extraction by mechanically scraping cells from MYA plates into a 50 mL falcon tube, yielding a suspension of adherant and floating cells in seawater. The suspensions were briefly vortexed to homogenise. One mL of each N. perurans cell suspension was added to 600 μL of RLT lysis buffer followed by vortexing for 10 min. Fresh lysozyme dilution (20 μL of 10 mg/mL) was added to each tube, prior to 30 min incubation at 37 °C on a shaker platform. Subsequently, 10 μL of Proteinase K (> 600 mAU/ml; QIAGEN, Hilden, Germany) was added into each tube followed by 30 min incubation at room temperature. The rest of the DNA extraction was performed following manufacturer’s instructions (AllPrep DNA/RNA Mini Kit [QIAGEN, Hilden, Germany]). All DNA concentrations after the extraction processing were measured using Qubit dsDNA BR Assay Kit (ThermoFisher Scientific, Paisley, United Kingdom), following its instructions.

Dhufaigh K.N., Dillon E., Botwright N., Birlanga V., Talbot A., O’Connor I., MacCarthy E, & Slattery O. (2021). Comparative proteomic profiling of newly acquired, virulent and attenuated Neoparamoeba perurans proteins associated with amoebic gill disease. Scientific Reports, 11, 6830.

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Optimized Intestinal Microbiome Sampling

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Fresh stool samples were collected on site from participants at baseline (prior to bowel cleansing and treatment), as well as at 6 weeks, 12 weeks, and 26 weeks post-treatment. A 200 mg aliquot was taken from the middle section of the stool and transferred to a 2-ml LoBind DNA tube for temporary storage at −80°C. In addition, FMT capsules from each batch of donations were reserved for microbiome assessment. Nucleic acid extraction was performed within 5 days of stool collection using a modified protocol of the AllPrep DNA/RNA Mini Kit (Qiagen, USA) [25 (link)]. Firstly, stool aliquots were incubated in 100 μl of lysis buffer (30 mM Tris-HCl, 1 mM EDTA, 15 mg/ml lysozyme) for 10 min at room temperature with regular agitation. Samples were then mixed with 1.2 ml RLT plus buffer (Qiagen, USA), 12 μl beta-mercaptoethanol (Sigma-Aldrich, USA), and 1 ml of acid-washed glass beads (≤106 μm, −140 US sieve; Sigma Aldrich, USA) and shaken vigorously at 30 Hz for 10 min on a TissueLyser II (Qiagen, USA). The homogenate was then passed through a QIAshredder spin column (Qiagen, USA), before continuing on with the standard AllPrep DNA/RNA Mini Kit protocol (Qiagen, USA) eluting in 100 μl of EB buffer. DNA purity was assessed using a NanoPhotometer N60 (Implen GmbH, Germany) and quantified by Qubit dsDNA Broad Range Assay (Thermo Fisher Scientific, USA).

Wilson B.C., Vatanen T., Jayasinghe T.N., Leong K.S., Derraik J.G., Albert B.B., Chiavaroli V., Svirskis D.M., Beck K.L., Conlon C.A., Jiang Y., Schierding W., Holland D.J., Cutfield W.S, & O’Sullivan J.M. (2021). Strain engraftment competition and functional augmentation in a multi-donor fecal microbiota transplantation trial for obesity. Microbiome, 9, 107.

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Isolation and Characterization of Leukemic and Healthy Cells

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Mononuclear cells from the ALL patients (P1-P4) were collected from bone marrow aspirates by Ficoll Isopaque (Pharmacia) densitygradient centrifugation at the time of diagnosis. The leukemic cell content of the samples exceeded 90% of the total cell con tent. DNA and total RNA were extracted from the ALL patient samples using reagents from the AllPrep DNA/RNA Mini Kit (QIAGEN). CD3 + T cells and CD19 + B cells were isolated from peripheral blood from healthy Swedish blood donors by positive selec tion using CD19 + and CD3 + Microbeads and MACS cell separation reagents (Miltenyi). DNA and total RNA was extracted from the cells by the AllPrep DNA/RNA Mini Kit (QIAGEN). RNA concentra tions were determined using Qubit (ThermoFisher) and the integrity of the RNA was verified by Agilent Bioanalyzer 2100 (Agilent technologies).

Wahlberg P., Lundmark A., Nordlund J., Busche S., Raine A., Tandre K., Rönnblom L., Sinnett D., Forestier E., Pastinen T., Lönnerholm G, & Syvänen A.C. (2016). DNA methylome analysis of acute lymphoblastic leukemia cells reveals stochastic de novo DNA methylation in CpG islands. Epigenomics, 8(10).

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Comprehensive Stool DNA/RNA Extraction

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DNA and RNA extractions were performed at the Liggins Institute (University of Auckland, New Zealand) using a modified protocol of the AllPrep DNA/RNA Mini Kit (#80204, Qiagen, Germany) as described previously^{19 (link)}. Briefly, stool aliquots were thawed and incubated in 100 µl of lysis buffer (30 mM Tris–HCl, 1 mM EDTA, 15 mg/ml lysozyme) and 10 µl proteinase K for 10 min at room temperature with regular agitation. Samples were then mixed with 1.2 ml RLT plus buffer (Qiagen, Germany), 12 µl beta-mercaptoethanol, and 1 ml of acid washed glass beads (≤ 106 µm, − 140 U.S. sieve; #G4649-100G, Sigma Aldrich, USA) and shaken vigorously at 30 Hz for 10 min on a TissueLyser II (Qiagen, Germany). The homogenate was then passed through a QIAshredder spin column (#79654, Qiagen, Germany), before continuing with the standard AllPrep DNA/RNA Mini Kit protocol (#80204, Qiagen, Germany). DNA was eluted in 75 µl of EB buffer and RNA eluted in 50 µl RNase free water. DNA was quantified by Qubit dsDNA High Sensitivity Assay Kit (#Q32851, Invitrogen, California, USA). RNA was quantified by Qubit RNA High Sensitivity Assay Kit (#Q32852, Invitrogen, California, USA) and quality assessed using Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer instrument (Agilent technologies, California, USA). Sample data (including RNA integrity Number, RIN) is available in Table S1.

Vatanen T., Sakwinska O., Wilson B., Combremont S., Cutfield W.S., Chan S.Y., Godfrey K.M, & O’Sullivan J.M. (2022). Transcription shifts in gut bacteria shared between mothers and their infants. Scientific Reports, 12, 1276.

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Targeted Genomic Sequencing of Rpe65 in Mice

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Genomic DNA (gDNA) from cultured cells or mouse RPE tissue was isolated using the DNeasy Blood and Tissue Kit (Qiagen, no. 69504) or AllPrep DNA/RNA Mini Kit (Qiagen, no. 80284). Total RNA was extracted from the mouse posterior eye cup using AllPrep DNA/RNA Mini Kit (Qiagen, no. 80284). Complementary DNA (cDNA) was synthesized from the total RNA using Superscript III first-strand synthesis SuperMix (Thermo Fisher Scientific, no. 18080400). From gDNA and cDNA templates, 200–300 bp PCR amplicons of on- and off-target predicted sites for Rpe65 were generated using primers with partial Illumina adapter sequences and then purified using the QIAquick PCR Purification Kit (Qiagen, no. 28106). Samples were sequenced on an Illumina MiSeq. Between 70,000 and 100,000 NGS reads for each sample were generated on single-end (1 × 150 bp) or paired-end (2 × 250 bp) runs. Primers used for off-target analysis are listed in Table S2.

Choi E.H., Suh S., Foik A.T., Leinonen H., Newby G.A., Gao X.D., Banskota S., Hoang T., Du S.W., Dong Z., Raguram A., Kohli S., Blackshaw S., Lyon D.C., Liu D.R, & Palczewski K. (2022). In vivo base editing rescues cone photoreceptors in a mouse model of early-onset inherited retinal degeneration. Nature Communications, 13, 1830.

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Comprehensive RNA and DNA Analysis

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Total RNA was extracted using an AllPrep DNA/RNA minikit (Qiagen, Hilden, Germany). RNA reverse transcription to cDNA was carried out using RevertAid H Minus Moloney murine leukemia virus reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s protocol. Quantitative PCR (qPCR) was performed using a MesaGreen qPCR MasterMix Plus for SYBR assay (Eurogentec, Seraing, Belgium). For each primer set, the qPCR was performed in duplicate, and the mRNA levels obtained were normalized to the average mRNA levels of three housekeeping genes (ß-globin, ß-actin, and GAPDH) measured in the same samples. For each PCR, a sample in which the DNA template was replaced with PCR-grade water was included as a negative control. To measure the EBV genome copy number per cell, total DNA was extracted using an AllPrep DNA/RNA minikit (Qiagen) and measured using a NanoDrop spectrophotometer (Thermo scientific, Waltham, MA, USA). Similar amounts of DNA were used as a template for TaqMan PCR, performed according to the protocol described by Accardi et al. [33 (link)]. The PCR primer sequences are indicated in Table 1. All the primers used for the first time in the present study were assessed for their efficiency (90% to 110%).

Manara F., Jay A., Odongo G.A., Mure F., Maroui M.A., Diederichs A., Sirand C., Cuenin C., Granai M., Mundo L., Hernandez-Vargas H., Lazzi S., Khoueiry R., Gruffat H., Herceg Z, & Accardi R. (2022). Epigenetic Alteration of the Cancer-Related Gene TGFBI in B Cells Infected with Epstein–Barr Virus and Exposed to Aflatoxin B1: Potential Role in Burkitt Lymphoma Development. Cancers, 14(5), 1284.

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Multimodal Nucleic Acid Extraction

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We used the AllPrep DNA/RNA mini kit (QIAGEN) to extract both DNA and RNA from organoid samples. Organoids were extracted from matrigel domes using Cell Recovery Solution (Corning, #354253) before adding lysis buffer.
Snap-frozen tumor tissues were used for RNA extraction. Tumor tissues were homogenized [Ultra-Turrax T25 homogenizer (Janke & Kunkel)], and lysed simultaneously. RNA was extracted using the AllPrep DNA/RNA mini kit (QIAGEN).
Paraffin-embedded primary tumor tissue blocks were used for DNA extraction. Five 10-μm-thick FFPE tissue sections were deparaffinized and hematoxylin-stained. Tumor areas were selected and microdissected manually into 5% Chelex 100 Resin (Bio-Rad) and cell lysis solution (Promega), using sterile scalpels. DNA was extracted by proteinase K (Roche) digestion by overnight incubation at 56°C.

Farshadi E.A., Chang J., Sampadi B., Doukas M., Van 't Land F., van der Sijde F., Vietsch E.E., Pothof J., Koerkamp B.G, & van Eijck C.H. (2021). Organoids Derived from Neoadjuvant FOLFIRINOX Patients Recapitulate Therapy Resistance in Pancreatic Ductal Adenocarcinoma. Clinical Cancer Research, 27(23), 6602-6612.

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Purification of Total RNA and miRNA

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RNA was extracted using Qiagen AllPrep DNA/RNA Mini Kit (Cat. no. 80204, Qiagen, CA, USA) and a Qiagen supplementary protocol “Purification of total RNA containing miRNA from animal cells using the RNeasy Plus Mini Kit (cat. no. 74134). In brief, the Qiagen AllPrep DNA/RNA Mini Kit was first used to lyse the cells and homogenize the lysate. The DNA was then bound to the AllPrep DNA spin column following the vendor's instructions. The flow-through, after DNA binding, was used to isolate RNA (containing miRNA) using the Qiagen supplementary protocol and the Qiagen RNeasy Plus Mini Kit mentioned above. The quality of RNA was controlled by BioAnalyzer Small RNA method.

Kilpinen L., Parmar A., Greco D., Korhonen M., Lehenkari P., Saavalainen P, & Laitinen S. (2016). Expansion induced microRNA changes in bone marrow mesenchymal stromal cells reveals interplay between immune regulation and cell cycle. Aging (Albany NY), 8(11), 2799-2812.

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DNA Methylation and Gene Expression Analysis

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Bisulphite treatment of gDNA extracted from SVF and peripheral blood leukocytes (PBL) by the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany), was performed using the EZ DNA Methylation Kit (Zymo Research, CA, USA). Converted gDNA was amplified by PCR using specific primers for PTPRD-DMR. Bisulphite sequencing (BS) was performed as reported in [17] . Primers are shown in Supplementary Table 2. See Supplementary Methods for details.
Total RNA isolation & quantitative real-time PCR Total RNA was extracted from SVF and 3T3-L1 cells using the AllPrep DNA/RNA Mini Kit (Qiagen). Real-time RT-PCR was performed as described in [26] . Primers are shown in Supplementary Table 2. See Supplementary methods for details.

Parrillo L., Spinelli R., Longo M., Desiderio A., Mirra P., Nigro C., Fiory F., Hedjazifar S., Mutarelli M., Carissimo A., Formisano P., Miele C., Smith U., Raciti G.A, & Beguinot F. (2020). Altered PTPRD DNA methylation associates with restricted adipogenesis in healthy first-degree relatives of Type 2 diabetes subjects. Epigenomics, 12(10).

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