After humane euthanasia, mice were weighed and intestinal tissues were collected. Ileal segments <0.25 cm in length were excised and placed immediately in RNA stabilizer (DNA/RNA Shield; Zymo Research; Irvine, CA) and stored at −80 °C until RNA isolation. Small RNAs were extracted using miRNeasy Mini Kit (Qiagen; Germantown, MD) using a QIAzol-chloroform extraction protocol. Total RNA including small RNAs were further digested to removed residual DNA using DNAase 1 (Thermo Fisher Scientific; Waltham, MA) and Turbo DNAse I kit (Thermo Fisher Scientific; Waltham, MA). Total RNA was then quantified using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific; Waltham, MA) and the Qubit RNA BR Assay Kit (Thermo Fisher Scientific; Waltham, MA). Purity of the total RNA was assessed using a Nanodrop ND-1000 UV-Vis Spectrophotometer (NanoDrop; Wilmington, DE). Handling of tissue was carried out under Michigan State University Environmental Health and Safety under Animal Use Form number 02/14-030-00.
Turbo dnase 1 kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Turbo DNase I kit is a laboratory product designed to rapidly and efficiently degrade DNA. It contains a heat-labile DNase I enzyme that can be easily inactivated after use. The kit provides a simple and effective way to remove DNA contaminants from RNA samples or other applications requiring DNA removal.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Agilent bioanalzyer, by Agilent Technologies (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Truseq rna sample preparation kit, by Illumina (2 mentions)
Qubit rna br assay kit, by Thermo Fisher Scientific (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Dna rna shield, by Zymo Research (1 mentions)
Superscript 3 rt, by Thermo Fisher Scientific (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
9 protocols using turbo dnase 1 kit
1
Intestinal RNA Isolation and Quantification
2
Oxidative Stress Response in Klebsiella
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Before RNA isolation, wild-type Klebsiella pneumoniae MGH78578 and the K. pneumoniae MGH78578 ΔsoxS strain were grown until mid-exponential state (MEP) by following an earlier standardized protocol (22 (link)). MEP-grown bacterial cells were treated with paraquat (7.81 μM) for 30 min to generate oxidative stress conditions. RNA was then extracted from both oxidative stressed and MEP-grown (control) cells using the Qiagen RNeasy minikit by following the manufacturer’s guidelines. Contaminating DNA was removed from the RNA sample using the Turbo DNase I kit (Thermo Fischer Scientific). RNA was then quantified using both Qubit RNA broad-range assay and the NanoDrop device.
3
Bulk RNA-seq Differential Expression Analysis
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Total RNA was isolated using TRIzol reagent (15596026; Thermo Fisher Scientific, Waltham, MA, USA) and genomic DNA was removed with the TURBO DNase I kit (AM2238; Thermo Fisher Scientific, Waltham, MA, USA). RNA concentration and quality were assessed with the Agilent Bioanalzyer (Agilent, Santa Clara, CA). Library construction was prepared using the TruSeq RNA sample Preparation Kit (Illumina), following the manufacturer's protocol for RNA input quantity relative to RNA quality. Sequencing was performed on Illumina HiSeq2500 (Illumina, Santa Clara, CA) to generate 100-bp paired-end reads. DESeq2 R package was used to normalize the original read count matrix for a unified expression level and define the differential expression of genes (DEGs). If the criterion of |log2 (fold change)| > 1 and P < 0.05, we could consider it statistically significant. Principal Component Analysis (PCA) was then performed. We converted Ensembl ID (downloaded from http://asia.ensembl.org ) into gene names with Perl language. Heatmaps were created by the pheatmap R package. R software (version 4.0.5) and the limma R package were employed to deal with data. Images were generated by the ggplot2 R package. GO and KEGG enrichment was implemented on these upregulated DEGs with clusterProfiler R package.
4
Transcriptomic Response of K. pneumoniae to Stress
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Wild-type K. pneumoniae MGH78578 was grown to mid-exponential phase (MEP) at 37°C in Müeller-Hinton broth. Cells were then treated with paraquat at different concentrations (0, 3.905, 7.81, 200, and 500 μM) or similarly with tetracycline (0, 0.5, 10, 100, and 500 μg/ml) for 30 min, and RNA was then extracted. All assays were run in triplicate. Under all conditions, RNA was extracted using an Qiagen RNeasy minikit by following the manufacturer's guidelines. Any contaminating DNA was removed from the RNA sample using the Turbo DNase I kit (Thermo Fischer Scientific). Purified RNA was subsequently quantified using both Qubit RNA broad-range assay and NanoDrop device.
5
Oxidative stress response in Klebsiella
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Before RNA isolation, wild type Klebsiella pneumoniae MGH78578 and K. pneumoniae MGH78578 ΔsoxS were grown until mid-exponential state (MEP) following an earlier standardized protocol 22 (link) .
MEP grown bacterial cells were treated with paraquat (7.81 µM) for 30 minutes to generate oxidative stress conditions. RNA was then extracted from both oxidative stressed and MEP grown (control) cells using QIAGEN RNeasy Mini Kit following manufacturers guidelines. Contaminating DNA was removed from the RNA sample using the Turbo DNase I kit (Thermo Fischer Scientific). RNA was then quantified using both Qubit RNA Broad Range Assay and the Nanodrop.
MEP grown bacterial cells were treated with paraquat (7.81 µM) for 30 minutes to generate oxidative stress conditions. RNA was then extracted from both oxidative stressed and MEP grown (control) cells using QIAGEN RNeasy Mini Kit following manufacturers guidelines. Contaminating DNA was removed from the RNA sample using the Turbo DNase I kit (Thermo Fischer Scientific). RNA was then quantified using both Qubit RNA Broad Range Assay and the Nanodrop.
6
Paraquat Stress Response in K. pneumoniae
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Wild type K. pneumoniae MGH78578 was grown to Mid Exponential Phase (MEP) at 37ºC in Müeller-Hinton Broth, cells were then treated with paraquat at different concentrations (0, 3.905, 7.81, 200 and extracted. All assays were run in triplicate. In all conditions, RNA was extracted using QIAGEN RNeasy Mini Kit following manufacturers guidelines. Any contaminating DNA was removed from the RNA sample using the Turbo DNase I kit (Thermo Fischer Scientific). Purified RNA was subsequently quantified using both Qubit RNA Broad Range Assay and Nanodrop.
7
RNA Isolation and RNA-seq Sample Preparation
8
Quantifying Schistosome Acetylcholinesterase Genes
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The level of expression of both the SmAChE1 and the SmTAChE gene in different life stages of the parasite, and in parasites treated with gene-specific siRNAs, was measured by RT-qPCR, using custom TaqMan gene expression systems (Applied Biosystems, CA). First, RNA from different life stages was isolated using TRIzol Reagent according to the manufacturer’s instructions (Invitrogen). RNA was then treated with DNaseI to remove any genomic DNA, using a Turbo-DNase I kit (Ambion). cDNA was synthesized using 0.5 µg RNA, an oligo (dT)12-18 primer and Superscript III RT (Invitrogen). For developmental expression, triose phosphate isomerase (TPI) was used as a reference gene, as previously (31 (link)). Primer sets and reporter probes labeled with 6-carboxyfluorescein (FAM) were obtained from Applied Biosystems, CA (Table S2
9
RNA Extraction from Flower Buds
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RNA was isolated from unopened flower buds of 2 mm from the indicated genotype and treatments using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions. Plants were grown in three separate batches with several buds taken from different plants from each batch representing the three replicates used in the RNA-seq for each genotype and temperature treatment. RNA was DNase treated using the Turbo DNase I kit (Ambion) according to the manual. RNA integrity was assayed via agarose gel electrophoresis and quantified using a Nanodrop.
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