Total RNA from 10 × 106 bone marrow cells was isolated using RNeasy Mini Kit (QIAGEN, Hilden, Germany). DNase treatment was applied using DNA-free DNase Treatment and Removal Reagents (Ambion, Austin, TX, USA). cDNA was synthesized from 1 µg of RNA using TaqMan Reverse Transcription Reagents (Thermo Fisher Scientific, Waltham, MA, USA) and oligo(dT) primers in a final volume of 25 μL. Real-time quantitative PCR reactions were performed in technical triplicates in 20 μL volume using AceQ SYBR qPCR Master Mix (Vazyme Biotech Co., Nanjing, China) and the respective primers (Supplementary Table S1 ) using a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). Primer set efficiency was first tested in a standard curve experiment. Relative expression was calculated from Ct obtained from Applied Biosystems ViiA 7 Real-Time PCR System software (Thermo Fisher Scientific). Since test and housekeeping primer set efficiencies differed >10%, we applied the corrective formula described in [40 (link)] to calculate the relative expression ratio. β-actin or Hprt expression was used for normalization (Supplementary Table S1 ).
Dnase treatment and removal reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
DNase Treatment and Removal Reagents are lab equipment designed to remove or inactivate DNase enzymes from samples. These reagents are used to prevent DNA degradation during various molecular biology workflows.
Automatically generated - may contain errors
Lab products found in correlation
Rnasin ribonuclease inhibitor, by Promega (3 mentions)
Aceq sybr qpcr master mix, by Vazyme (2 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Taqman reverse transcription reagent, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Taqman gene expression assay for influenza matrix, by Thermo Fisher Scientific (2 mentions)
Abi 7500 real time pcr system platform, by Thermo Fisher Scientific (2 mentions)
Sequence detections software version 1, by Thermo Fisher Scientific (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Nanodrop technology, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
M mlv rt, by Promega (2 mentions)
Platinum sybr green qpcr supermix udg, by Thermo Fisher Scientific (2 mentions)
Dna engine opticon 2 continuous fluorescence detection system, by Bio-Rad (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
High pure isolation kit, by Roche (1 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (1 mentions)
Nanophotometer, by Implen (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
14 protocols using dnase treatment and removal reagent
1
Quantitative RNA Expression Analysis
2
Transcriptomic Analysis of Labrenzia sp. PHM005 Pederin Cluster
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Mutant strain PHM005ΔPKS4 was cultured in MBM + vit medium and the total RNA was extracted by High-pure Isolation Kit (Roche) after 24 h. Extracted RNA was additionally treated by DNA-free DNaseTreatment and Removal Reagents (Ambion). The RNA concentration was measured by using a NanoPhotometer (Implen). Reverse transcription of 1 μg of DNA-free RNA per sample was done using the Transcriptor First Strand cDNA Synthesis Kit (Roche) to generate cDNA and RT-PCR semi-quantitative analyses were carried out to check the mRNA levels. The rpoD gene was used as the housekeeping gene. Primers for RT–PCR for Labrenzia sp. PHM005 pederin cluster genes, as well as the housekeeping rpoD gene, were designed using Primer3 implemented by Geneious v.10.0.2 (Supplementary Table S2 ). Relative mRNA expression was visualized in 1.5% agarose gel. Negative controls contained RNA samples without the reverse transcriptase. The RNA extraction from the strain PHM005 for the transcriptomics analysis was performed in the early exponential phase (12 h) as described previously. Transcriptomics and bioinformatics analyses were carried out by Vertis Biotechnologie AG. The percentage of the mapped reads to annotated genes is 78.4, 80.8, and 77.0% for each replicate sample. The results represent a mean of three replicates.
3
Quantitative RT-PCR for Influenza Virus
4
Quantitative RT-PCR for Influenza Virus
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Quantitative RT-PCR was performed to assess viral mRNA transcripts from infected ferret tracheal and lung samples used for sequencing. Total RNAs were treated with DNase using DNA-free DNase Treatment and Removal Reagents (Ambion, Inc, Austin, TX). cDNAs from total RNAs were generated using the QuantiTect reverse transcription kit (Qiagen Inc.). A custom-designed TaqMan gene expression assay for influenza Matrix (M) sequence (MPCONS2010), with primers that have complete homology with both 1918 and CA04 M sequences, was ordered from Applied Biosystems, Inc. Taqman experiments were performed on the ABI 7500 Real-Time PCR System platform and each sample was run in quadruplicate. Ribosomal RNA (18S) was used as endogenous control to normalize quantification of each target within tissues using Applied Biosystems Sequence Detections Software version 1.3. The relative amount of viral mRNA (log 10) is presented in the final results.
5
RNA Extraction from Hippocampal Samples
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Isolated hippocampi were dissociated, preserved in RNAlater, and total RNA was isolated from each sample using the RNeasy Kit (Qiagen), as described previously.20 (link) Briefly, eluates were DNase-treated using the DNA-free Kit (DNase Treatment and Removal Reagents; Ambion) and RNA quality/purity was quantitated using spectrophotometric analysis at OD260/OD280.
6
RNA Extraction and Reverse Transcription
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Total RNA was extracted from human adipose tissue and adipocyte cultures on the 12th day of differentiation using RNeasy Lipid Tissue and RNeasy Mini Kit (Qiagen GmbH, Germany). Extracted RNA was quantified using NanoDrop technology (Fisher Scientific SAS, Illkirch Cedex, France) and immediately stored at −80 °C until needed. For DNAse treatment and reverse transcription, RNA (2 μg) was treated with DNase treatment and removal reagents (Ambion, Inc., Austin, TX, USA) using the manufacturer’s instructions. DNAse-treated RNA was reverse-transcribed for 1 h at 37 °C with 150 ng random hexamers, 0.5 mM dNTPs, 20 U of RNAsin Ribonuclease Inhibitor, and 200 U of M-MLV Reverse Transcriptase (Promega, Madison, WI, USA).
7
Bacterial mRNA Enrichment Protocol
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Both the wt of P. pseudoalcaligenes CECT 5344 R1 and the fur− mutant were grown at 30°C in LB medium from three independent starter cultures. At the mid‐exponential phase (O.D.600 = 0.3), 10 ml of each sample was collected and harvested by centrifugation. Total RNA was isolated using the Aurum Total RNA Mini Kit (Bio‐Rad, Hercules, CA, USA), and the purified RNA was treated with ‘DNase Treatment and Removal’ reagents (Ambion, Foster City, CA, USA), according to the manufacturer's instructions. After DNase treatment, a MICROBExpress™ Bacterial mRNA Enrichment Kit (Ambion) was used to remove bacterial rRNA from the total RNA samples.
RNA was quantified using a NanoDrop1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the purity and integrity of RNA samples was measured using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip Kit (Agilent Technologies, Santa Clara, CA, USA). All samples displayed a 260/280 ratio > 2.0 and RNA integrity numbers ≥ 9.
RNA was quantified using a NanoDrop1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the purity and integrity of RNA samples was measured using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip Kit (Agilent Technologies, Santa Clara, CA, USA). All samples displayed a 260/280 ratio > 2.0 and RNA integrity numbers ≥ 9.
8
Quantifying gene expression using qPCR
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Total RNA from stably selected cell lines was extracted using TRIzol (Invitrogen) and treated with DNA-free DNase Treatment and Removal Reagents (Ambion, Austin, TX). High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) was used to convert total RNA to cDNA. Real-time PCR using TaqMan Universal PCR master mix was performed to analyze RNA expression levels with Applied Biosystems Standard Real-Time PCR systems. TaqMan gene expression assays of DCTN3 (Hs00989657_m1), STOML2 (Hs00203730_m1), VCP (Hs00997650_m1), and 18s rRNA (Hs99999901_s1) were purchased from Applied Biosystems. Triplicate reactions were performed for each sample and standard error was calculated using ABI software (Applied Biosystems, Foster City, CA). Relative expression values using the average of cycle thresholds of target genes and 18s rRNA were calculated using the 2−ΔΔCt method. A value of 1 was assigned to the control empty vector sample.
9
Quantitative Gene Expression Analysis
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Total RNA was extracted by RNeasy Mini Kit (QIAGEN, Hilden, Germany) from 10 × 106 bone marrow or spleen cells previously lysed in RLT buffer (QIAGEN) supplemented with 1% β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) and stored at −80 °C. RNA was treated with DNA-free DNase Treatment and Removal Reagents (Ambion, Austin, TX, USA). DNase-treated total RNA (1 µg) was reverse transcribed with oligo(dT) and TaqMan Reverse Transcription Reagents (Thermo Fisher Scientific, Waltham, MA, USA). The resulting cDNA was diluted tenfold before use. Real-time quantitative PCR was performed in technical triplicates using AceQ SYBR qPCR Master Mix (Vazyme Biotech Co., Nanjing, China) and the corresponding primers (Supplementary Table 1 ) using a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). All RT–qPCR analyses were performed on an Applied Biosystems QuantStudio Software V1.3 (Thermo Fisher Scientific). Primer set efficiency was firstly calculated in a standard curve experiment. Relative gene expression levels were calculated using the comparative CT method. Since target and housekeeping primer set efficiencies differed >10%, we applied the corrective formula described in [21 (link)] to calculate the relative expression ratio. Gene expression levels were then normalized to that of Hprt.
10
Total RNA Extraction and qPCR Analysis
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Total RNA was extracted using RNeasy Lipid or Mini Kits (Qiagen Inc., Valencia, CA, USA) for tissue and cell culture samples, respectively, following the supplier’s instructions. RNA content was quantified using NanoDrop technology (Fisher Scientific SAS, Illkirch Cedex, France) and quality checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA samples were treated with DNase Treatment and Removal Reagents (Thermo Fisher, Waltham, MA, USA) and reverse-transcribed for 1 h at 37 °C with 150-ng random primers, 0.5-mM dNTPs, 20 units of RNAsin Ribonuclease Inhibitor and 200 units of M-MLV RT (Promega, Madison, WI, USA). Primer sequences and amplification conditions are reported in Table 2 . Real-time PCR was carried out with Platinum® SYBR® Green qPCR SuperMix-UDG (Thermo Fisher) on a DNA Engine OpticonTM 2 Continuous Fluorescence Detection System (MJ Research, Waltham, MA, USA). All experiments were performed in duplicate. Samples (5 ng of cDNA) were normalized by 18S rRNA content and reported as an arbitrary unit (a.u.) ratio or as a fold increase/decrease with respect to the control.
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