Total RNA was collected from confluent T25 flasks using the mirVana kit (Ambion, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. RNA was treated with an RNAse-free DNase kit (Invitrogen, USA) to remove any residual DNA and reverse transcribed using an oligo dT primer and reverse transcription kit (Invitrogen). The negative control (-RT) was split away and was subjected to the same treatment but without the addition of reverse transcriptase. Genomic DNA served as a positive control and was isolated using the RNeasy Plant Mini Kit (Qiagen, Germany). The sRNA enriched from each of the stable cell lines was isolated using a mirVana kit (Ambion, Thermo Fisher Scientific, USA) following the manufacturer’s instructions. Primers for RT-PCR were designed as follows:
Rnase free dnase kit
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
The RNAse-free DNAse kit is a tool designed to remove DNA contamination from RNA samples. It provides a reliable and efficient method for the digestion of DNA to ensure the integrity and purity of RNA for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Nanodrop readings, by Thermo Fisher Scientific (3 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (3 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Trizol extraction chemical, by Thermo Fisher Scientific (2 mentions)
Trizol extraction reagent, by Thermo Fisher Scientific (2 mentions)
Mirvana kit, by Thermo Fisher Scientific (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Rt master mix, by Applied Biological Materials (1 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (1 mentions)
Applied biosystems quantstudio 6, by Thermo Fisher Scientific (1 mentions)
Feature extraction software, by Agilent Technologies (1 mentions)
Microarray scanner system, by Agilent Technologies (1 mentions)
Agilent low input quick amp labeling kit, by Agilent Technologies (1 mentions)
Sureprint g3 human ge 8 60 k microarrays, by Agilent Technologies (1 mentions)
Kapa library quantification kit, by Roche (1 mentions)
Hiseq x10, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
19 protocols using rnase free dnase kit
1
Total RNA Extraction and miRNA Enrichment
2
Transcriptomic Analysis of Low-Temperature Adaptation in Maize
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Genes with well annotation involving in low-temperature adaption were collected from Oryza sativa, Arabidopsis thaliana, and Sorghum bicolor, and performed BLAST analysis in MaizeGDB1 . Six genes located in our confident QTL interval were selected for quantitative PCR (qPCR) validation.
Two groups of seedlings growing in paper rolls were cultivated under 10°C and 25°C germination conditions, respectively, shoots were collected for RNA isolation once they have similar seedling length at 15 DAS (grown under 10°C) and 3 DAS (grown under 25°C), respectively. Ten shoots in each replication were pooled and grinded in liquid nitrogen for total RNA extraction using RNAprep pure Plant Kit [Tiangen Biotech (Beijing)]. RNA samples were treated with RNase-free DNase Kit (Invitrogen) to remove DNA contamination. Followed by reverse transcription reaction of cDNA with RT MasterMix (Applied Biological Materials Inc.), qPCR analysis was performed on Applied Biosystems QuantStudio 6 (Thermo Fisher) using qPCR MasterMix solution (Applied Biological Materials Inc.). The primers used in qPCR were listed in Supplementary TableS2 . The maize ZmGAPDH gene was used as an internal control (Gu et al., 2013 (link)). The mean value from three replications was used as final gene expression.
Two groups of seedlings growing in paper rolls were cultivated under 10°C and 25°C germination conditions, respectively, shoots were collected for RNA isolation once they have similar seedling length at 15 DAS (grown under 10°C) and 3 DAS (grown under 25°C), respectively. Ten shoots in each replication were pooled and grinded in liquid nitrogen for total RNA extraction using RNAprep pure Plant Kit [Tiangen Biotech (Beijing)]. RNA samples were treated with RNase-free DNase Kit (Invitrogen) to remove DNA contamination. Followed by reverse transcription reaction of cDNA with RT MasterMix (Applied Biological Materials Inc.), qPCR analysis was performed on Applied Biosystems QuantStudio 6 (Thermo Fisher) using qPCR MasterMix solution (Applied Biological Materials Inc.). The primers used in qPCR were listed in Supplementary Table
3
Transcriptome Analysis of Treated Samples
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Total RNA was prepared with Trizol reagent (Invitrogen) according to the manufacturer’s instructions. To avoid genomic DNA contamination, total RNA samples were treated with a RNase-free DNase Kit (Invitrogen) following the manufacturer’s instructions.
The purified total RNA was used to produce Cy3-labeled cDNA using Agilent Low Input Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer’s instructions. Agilent SurePrint G3 Human GE 8 × 60 k microarrays were used, and the hybridization was performed according to a previous published protocol53 (link). Microarrays were scanned with an Agilent’s Microarray Scanner System, and the resulting images were analyzed with the Agilent Feature Extraction software. Genes were considered differentially expressed with a 2-fold change in the treated samples compared to the control sample.
The purified total RNA was used to produce Cy3-labeled cDNA using Agilent Low Input Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer’s instructions. Agilent SurePrint G3 Human GE 8 × 60 k microarrays were used, and the hybridization was performed according to a previous published protocol53 (link). Microarrays were scanned with an Agilent’s Microarray Scanner System, and the resulting images were analyzed with the Agilent Feature Extraction software. Genes were considered differentially expressed with a 2-fold change in the treated samples compared to the control sample.
4
Comprehensive Biochemical and Molecular Analyses
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Kits for Transaminases (ALT and AST), creatinine, urea, cholesterol (Chol), triglycerides (TriG), low- and high-density lipoprotein (LDL, HDL), total protein (TP), and albumin (Alb) were supplied by Randox Co, (Antrim, UK). Kits for nitric oxide (NO), malondialdehyde (MDA), glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) were purchased from Eagle diagnostics (Dallas, TX, USA). Kits for carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and tumor necrosis factor-alpha (TNF-α) were provided by BiochemImmuno Systems Co. (Montreal, Canada). Kit for first-strand cDNA synthesis was obtained from iNtRON Biotechnology (Seoul, Korea). SYBR® Premix Ex TaqTM kit was supplied by TaKaRa Biotech. Co. Ltd. (Shiga, Japan). TRIzol® reagent and RNAse-free DNAse kit were purchased from Invitrogen (Germany).
5
Liver Transcriptome Analysis by RNA-seq
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The aliquots of livers from three randomly selected rats from the normal diet, HCHFD, and HCHFD+HTE groups were ground into powder in liquid nitrogen. Total RNA was extracted from using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. To avoid genomic DNA contamination, total RNA samples were treated with a RNase-Free DNase Kit (Invitrogen) following the manufacturer’s instructions. The quality of RNA was evaluated using Agilent 2100 BioAnalyzer (Agilent Technologies, Palo Alto, CA, USA). The purified RNA then was used to synthesize double-strand complementary DNA (cDNA). The cDNA libraries were quantified using the Kapa Library Quantification Kit (Kapa Biosystems, Boston, MA, USA). After cluster amplification of the denatured templates, samples in flow cells were sequenced using the Illumina HiSeq X10 (Illumina) with the strategy of PE50.
StringTie (version 1.3.3b) was used to align transcript sequences obtained from RNA-seq to the UCSC reference genome rn6 and to estimate the transcript levels of genes. Differentially expressed genes were identified using DESeq2 (version 1.16.1) with the cutoff at fold change ≥2 and p value ≤0.05.
StringTie (version 1.3.3b) was used to align transcript sequences obtained from RNA-seq to the UCSC reference genome rn6 and to estimate the transcript levels of genes. Differentially expressed genes were identified using DESeq2 (version 1.16.1) with the cutoff at fold change ≥2 and p value ≤0.05.
6
RNA Sequencing of K. pneumoniae
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The isolates were grown in high osmolarity LB broth at 37°C till it reached the logarithmic phase and the RNA was extracted using the RNeasy® mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol. DNA contamination was eliminated using an RNase-free DNase Kit (Invitrogen). The concentration of RNA was measured using a Nanodrop™ spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA sequencing was performed using a HiSeq2500 sequencer (Illumina, San Diego, CA, USA) and transcriptomic analysis was carried out using CLRNASeq software (http://www.chunlab.com/software_clrnaseq_download ; Chunlab, Seoul, Korea). The data were normalized using the reads per kilobase per million mapped reads (RPKM), relative log expression (RLE), and trimmed mean of M-value (TMM) methods. K. pneumoniae ATCC 13883 was used as the reference strain (Assembly ID: GCA_000742135.1).
7
BTKi Treatment Effects on MCL Transcriptome
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MCL cells were treated with or without BTKi (2 μM) for 48 h, and total RNA was extracted with TRIzol (#15596018; Thermo Fisher Scientific, Beijing) and RNA samples that meet following requirements were used in subsequent experiments: RNA integrity number (RIN) > 7.0 and a 28S:18S ratio > 1.8. To avoid genomic DNA contamination, total RNA samples were treated with a RNase-Free DNase Kit (Invitrogen) following manufacturer’s instructions and cDNA was synthesized from 2 μg of total RNA using Superscript III First-strand Synthesis System (#18080–051; Invitrogen, Beijing) according to manufacturer’s instructions. qRT-PCR was performed using PowerUp SYBR Green Mix (#00710493; Applied Biosystems, Beijing). The primers used for qRT-PCR were shown in Additional file 2 : supplementary Table S1.
8
Total RNA Isolation from Cultured Cells
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Trizol reagent extraction method (Invitrogen, Germany, catalog no. 15596-026) was used to isolate total RNA from the cultivated cells. The isolated RNA pellets were treated with RNAse-free DNAse kit (Invitrogen, Germany). The RNA concentrations (ng/µL) and purities for all aliquots were determined using NanoDrop® 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The isolated RNA aliquots were kept at −80 °C till the reverse transcription step.
9
Genomic and Transcriptomic Analysis of Clinical Strains
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For genomic sequence analysis of two clinical strains, genomic DNA was extracted using a Wizard genomic DNA purification kit (Promega, Madison, WI, USA). Library preparation was carried out using a TruSeq Nano DNA sample preparation kit (Illumina, San Diego, CA, USA) per the manufacturer's protocol, and the sequencing was carried out by using an Illumina-MiSeq sequencing platform (2× 300-bp paired-end protocol).
For gene expression analysis, we isolated RNA by growing the clinical isolates to the logarithmic phase in high-osmolality LB broth at 37°C and extracting the RNA using an RNeasy minikit (Qiagen GmbH, Hilden, Germany). On-column DNA digestion was carried out using an RNase-free DNase kit (Invitrogen, Carlsbad, CA, USA). RNA concentrations were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing library preparation was carried out using a TruSeq stranded total RNA library preparation kit (Illumina, San Diego, CA, USA). We performed RNA sequencing for two biological replicates using an Illumina NextSeq 500 sequencer (Illumina, San Diego, CA, USA) with ∼25 million paired-end reads.
For gene expression analysis, we isolated RNA by growing the clinical isolates to the logarithmic phase in high-osmolality LB broth at 37°C and extracting the RNA using an RNeasy minikit (Qiagen GmbH, Hilden, Germany). On-column DNA digestion was carried out using an RNase-free DNase kit (Invitrogen, Carlsbad, CA, USA). RNA concentrations were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing library preparation was carried out using a TruSeq stranded total RNA library preparation kit (Illumina, San Diego, CA, USA). We performed RNA sequencing for two biological replicates using an Illumina NextSeq 500 sequencer (Illumina, San Diego, CA, USA) with ∼25 million paired-end reads.
10
Cardiac Tissue RNA Extraction and cDNA Synthesis
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TRIzol® extraction Chemical (Invitrogen) was used to isolate the total genomic RNA of the cardiac tissues of all treated animals. RNA pellet was stored in DEPC treated water after completion of the isolation procedures. The pellet of isolated RNA was treated with RNAse-free DNAse kit (Invitrogen, Germany) to digest the potential DNA residues. RNA aliquots were placed at −20 °C or utilized immediately for reverse transcription [20 (link)].
First Strand cDNA Synthesis Kit (RevertAidTM, MBI Fermentas) was utilized for the synthesis of the cDNA copy from the cardiac tissues via reverse transcription reaction. A reverse transcription reaction program of 25 °C for 10 min, then one hour at 42 °C then 5 min at 95 °C was employed to get the cDNA copy of the genome. Eventually, tubes of reaction containing cDNA copy were collected on ice until utilization for cDNA amplification [21 ].
First Strand cDNA Synthesis Kit (RevertAidTM, MBI Fermentas) was utilized for the synthesis of the cDNA copy from the cardiac tissues via reverse transcription reaction. A reverse transcription reaction program of 25 °C for 10 min, then one hour at 42 °C then 5 min at 95 °C was employed to get the cDNA copy of the genome. Eventually, tubes of reaction containing cDNA copy were collected on ice until utilization for cDNA amplification [21 ].
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