Bacterial cultures from S or P conditions were harvested at the mid-log phase of growth (8h for S and 4h for P) and diluted to an OD600 of 0.6. Then, 1 ml of the culture was taken and mixed with twice the volume of RNAprotect reagent (Qiagen) and incubated at room temperature for 10 min. Total RNA was extracted using a Qiagen RNeasy minikit with an On-column DNase digestion kit per the manufacturer’s instructions. Reverse transcription was performed using an Invitrogen SuperScrip II Reverse Transcriptase kit from 50 ng of total RNA. Real-time PCR (RT-PCR) was carried out using a Power SYBR Green Master Mix (Thermo Fisher). Reactions were performed in technical triplicates. The 20 μl reaction mixture contained 100 ng of total RNA, 10 μl green reaction mix and 100 nM each of forward and reverse primers for specific targets (See Table S2 ). Threshold values were calculated using QuantStudio 3 software (Applied Biosystems) and used to determine fold changes between samples. Data were normalized to the gyrA expression levels.
On column dnase digestion kit
Manufactured by Qiagen
Sourced in United States
The On-column DNase digestion kit is a laboratory product designed to remove DNA contamination from RNA samples during the RNA purification process. It provides a convenient method for on-column DNase treatment to ensure high-quality, DNA-free RNA for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (3 mentions)
Rnaprotect reagent, by Qiagen (2 mentions)
Direct zol rna miniprep kit, by Zymo Research (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Rna ultrasense one step quantitative rt pcr system, by Thermo Fisher Scientific (2 mentions)
Quantstudio 3, by Thermo Fisher Scientific (1 mentions)
Power sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Superscrip 2 reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Viia7 rt pcr system, by Thermo Fisher Scientific (1 mentions)
Quantstudio software, by Thermo Fisher Scientific (1 mentions)
Itaq universal sybr one step kit, by Bio-Rad (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Rneasy tissue kit, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Nextseq 500 platform, by Illumina (1 mentions)
Smart seq ultra low input rna kit, by Takara Bio (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Kapa sybr fast universal qpcr kit, by Merck Group (1 mentions)
7 protocols using on column dnase digestion kit
1
Quantitative Real-Time PCR for Gene Expression
2
RNA Extraction and qRT-PCR Quantification
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Cells were lysed with TriZol LS, and total RNA was isolated from cells with the Direct-zol RNA mini prep kit (Zymo research) according to the manufacturer's protocol. RNA was treated with the On-Column DNase Digestion kit (Qiagen) according to the supplied protocol. The amount of the intracellular RNA was estimated by real-time quantitative PCR performed with the RNA UltraSense™ One-Step Quantitative RT-PCR System (Applied Biosystems) by using the following primer sets: CDC N1 (forward 5′-GAC CCC AAA ATC AGC GAA AT, reverse 5′-TCT GGT TAC TGC CAG TTG AAT CTG, probe 5′-FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1), 18S rRNA (TaqMan Gene expression assay from ThermoFisher, Hs99999901_s1), and GusB (TaqMan Gene expression assay from ThermoFisher, Hs99999908_m1).
3
Quantitative RNA Isolation and Analysis
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Cells were lysed with TriZol LS, and total RNA was isolated from cells with the Direct-zol RNA mini prep kit (Zymo research) according to the manufacturer’s protocol. For RNA sequencing experiments, RNA was treated with the On-Column DNase Digestion kit (Qiagen) according to the supplied protocol. The amount of the intracellular RNA was estimated by real-time quantitative PCR performed with the RNA UltraSense™ One-Step Quantitative RT-PCR System (Applied Biosystems) by using the following primer sets: CDC N1 (forward 5′- GAC CCC AAA ATC AGC GAA AT, reverse 5’- TCT GGT TAC TGC CAG TTG AAT CTG, probe 5’-FAM- ACC CCG CAT TAC GTT TGG TGG ACC -BHQ1), 18S rRNA (TaqMan Gene expression assay from ThermoFisher, Hs99999901_s1), and GusB (TaqMan Gene expression assay from ThermoFisher, Hs99999908_m1).
4
Gene Expression Analysis of Bacterial Growth
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Bacteria were grown under swimming, swarm agar, and hard agar conditions as detailed above, supplemented with 0.5% glucose (wt/vol). Cultures (in triplicate) were harvested after 4 h of growth, standardized to an OD600 of 0.5, with 1 ml taken and mixed with twice the volume of RNAprotect reagent (Qiagen) before incubation was performed at room temperature for 5 min. Samples were pelleted at 12,000 rpm for 10 min before the pellet was used in total RNA extraction using a Qiagen RNeasy minikit with an On-column DNase digestion kit per the manufacturer’s instructions.
Real-time PCR (RT-PCR) was carried out using an iTaq Universal SYBR one-step kit (Bio-Rad) together with a ViiA-7 RT-PCR system (Thermo Fisher). Reactions were performed in duplicate, and the reaction mixtures contained 50 ng of total RNA, 10 µl iTaq green reaction mix (2×), 0.25 µl iScript reverse transcriptase, 150 nM (each) forward and reverse primers for relevant targets (Table S1 ), and nuclease-free H2O used to reach a final reaction volume of 20 µl. Threshold values were calculated using QuantStudio software (Thermo Fisher) and used to determine fold changes between samples. Student's t test was used to calculate P values. Data were normalized to the expression of gyrA.
Real-time PCR (RT-PCR) was carried out using an iTaq Universal SYBR one-step kit (Bio-Rad) together with a ViiA-7 RT-PCR system (Thermo Fisher). Reactions were performed in duplicate, and the reaction mixtures contained 50 ng of total RNA, 10 µl iTaq green reaction mix (2×), 0.25 µl iScript reverse transcriptase, 150 nM (each) forward and reverse primers for relevant targets (
5
Isolation and Purification of High-Quality RNA from Lung Tissue
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Total RNA was isolated from frozen lung tissue using TRIzol reagent (Invitrogen, Life Technologies, Grand Island, NY, USA) according to manufacturer's protocol, then further purified with the RNeasy Tissue kit (Qiagen, Valencia, CA, USA), and the contaminant genomic DNA was removed with a Qiagen on-column DNase digestion kit. RNA concentration was measured with NanoDrop® ND1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA), and its integrity and purity were verified by Agilent 2100 Bioanalyzer Capillary Gel Electrophoresis System (Agilent, Palo Alto, CA, USA).
6
Transcriptome Profiling of Human Adipose Tissue
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Total RNA was extracted and purified from human fat specimens using a Qiagen RNeasy Micro kit (Qiagen), and residual genomic DNA was further removed by an on-column DNase digestion kit (Qiagen). Library construction, sequencing, and data analysis were performed at the Center for Cancer Computational Biology Core Facilities at Dana-Farber Cancer Institute (DFCI). Sequencing libraries were prepared using a SMART-Seq Ultra Low Input RNA kit (Clontech). The resulting library size distributions were analyzed using a Bioanalyzer (Agilent). The concentration of the library was determined using a DNA High-Sensitivity Qubit assay, and the final functional library concentration was determined through qPCR using Illumina adaptor-specific primers with a KAPA SYBR FAST Universal qPCR kit (Sigma–Aldrich).The library pools were loaded at final concentrations of 2 pM on single-read 75 flow cells and sequenced on an Illumina NextSeq 500 platform. Sequencing reads were aligned to the reference genome (Ensembl GRCh37.75) using the RNA-specific STAR aligner (v2.3.1z4).
7
Transcriptome Analysis of L. racemosus Roots
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Control-, salt- and ammonium-treated root tissues of L. racemosus were used for RNA-sequencing. Total RNA was extracted using RNeasy mini kit with the inclusion of an on-column DNase digestion kit (Qiagen). Using the isolated RNA, mRNA-seq libraries were constructed for the three conditions using TruSeq RNA Sample Preparation. To generate 150-bp pair-end reads, the libraries were sequenced by HiSeq2500 according to the standard protocol.
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