After obtaining informed consent, vaginal swabs were prospectively collected daily for 10 wk from 135 women (Ravel et al. 2013 (link)). Two samples were from each of two women who developed vaginal candidiasis prior to the development of symptoms and at the time of symptoms but prior to treatment. The swabs were immediately stored in RNAlater (Qiagen) after collection and stored at −20°C for a week and then at −80°C until RNA extraction. To isolate total RNA, the cells were rinsed from the swabs with ice-cold PBS, incubated with acid phenol, and lysed by bead beating. The nucleic acids were purified chloroform-isoamyl alcohol and ethanol precipitated. The samples were treated with the TURBO DNA-free DNase Treatment kit (Ambion), after which RNA was purified using the RNeasy Mini Kit. Samples are enriched for mRNA using a combination of the Ribo-Zero rRNA Removal Kits for gram-negative, gram-positive, and human/mouse/rat RNA (Epicentre Biotechnologies). Absence of the DNA contamination in these samples was verified by PCR using 16S rRNA primers. The clinical study protocol was approved by the Institutional Review Board of the University of Alabama at Birmingham and the University of Maryland School of Medicine. Written informed consent was appropriately obtained from all participants.
Turbo dna free dnase treatment kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Turbo DNA-free DNAse Treatment Kit is a tool designed to remove contaminating DNA from RNA samples. It utilizes a recombinant DNase I enzyme to efficiently degrade double-stranded and single-stranded DNA, without affecting the integrity of the RNA.
Automatically generated - may contain errors
Lab products found in correlation
Totally rna kit, by Thermo Fisher Scientific (2 mentions)
Uracil dna glycosylase, by Roche (2 mentions)
Iscript dna synthesis kit, by Bio-Rad (2 mentions)
Faststart sybr green master mix, by Roche (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Thermo Fisher Scientific (1 mentions)
Rnalater, by Qiagen (1 mentions)
Rnaqueous kit, by Thermo Fisher Scientific (1 mentions)
Viia7 qrt pcr system, by Thermo Fisher Scientific (1 mentions)
Qubit rna hs assay, by Thermo Fisher Scientific (1 mentions)
Rneasy midi, by Qiagen (1 mentions)
Mini kits, by Qiagen (1 mentions)
Acid phenol chloroform ph 4, by Thermo Fisher Scientific (1 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
7 protocols using turbo dna free dnase treatment kit
1
Longitudinal Vaginal Microbiome Study
2
Quantitative Gene Expression Analysis
3
Gene Expression Quantitation by qPCR
4
Quantifying Gene Expression in Cardiac Ventricles
5
RNA Extraction and qPCR Analysis
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We used a modified protocol adapted from methods previously described for RNA extraction [43 (link)]. Total RNA was isolated from ipsilateral hippocampus using Trizol (Invitrogen, Carlsbad, CA) and DNase treated using TURBO DNA-free DNase Treatment Kit (Ambion, Austin, Tx). First-strand cDNA was synthesized using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Gene expression was assayed using the Power SYBR Green PCR Master Mix (Applied Biosystems) with primers listed below (Table 3 ) and quantified using the Step One Plus Real-Time PCR system (Applied Biosystems). Relative fold changes were calculated using the comparative threshold cycle methods (2-ΔΔCt).
qRT-PCR primers
| Genes | Forward primer | Reverse primer |
|---|---|---|
| qRT-PCR oligonucleotide | ||
| Il-6 | 5′-CAGAGGATACCACTACCAACAG-3’ | 5′-TCTCATTTCCACCACGATTTCCC-3’ |
| Il-10 | 5′-CAGGACTTTAAGGGTTAC-3’ | 5′-ATTTTCACAGGGGAGAATC-3’ |
| Il-16 | 5′-GAGGTGTGGTGATGAGATTG-3’ | 5′-GCTTACGATGATGGGAACTG-3’ |
| Il-17 | 5′-CAGCGATCATCCCTCAAAG-3’ | 5′-CGCCAAGGGAGTTAAAGAC-3’ |
| Bdnf | 5′-GAGCGTGTGTGACGTATTAG-3’ | 5′-CTTTGGATACCGGGACTTTC-3’ |
| Cxcl13 | 5′-CATAGATCGGATTCAAGTTACGCC-3’ | 5′-GTAACCATTTGGCACGAGGATTC-3’ |
| β-Actin | 5′-CTGCCTGACGGCCAAGTCATCAC-3’ | 5′-GTCAACGTCACACTTCATGATGG-3’ |
6
High-Yield RNA Extraction from Saccharolobus
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Saccharolobus islandicus RNA was extracted using RNeasy Midi or Mini kits (Qiagen, Hilden, Germany) with modifications based on published methods (Buetti-Dinh et al., 2016 (link)). Briefly, frozen biomass pellets were defrosted on wet-ice, resuspended in 1 ml lysis buffer, transferred to tubes containing 0.5 mm glass beads, then subjected to three cycles of freeze-thaw with bead beating to aid in cell lysis. We followed kit instructions for the remainder of the RNA extraction. For samples extracted with RNeasy Midi kits, we used our maximum centrifugation speed (3,163 × g) for all steps and increased centrifugation time of cell lysate through the filter column to 25 min and the Buffer RW1 wash step to 8 min.
Total RNA was treated with a TURBO DNA-free DNase treatment kit (Invitrogen, Waltham, MA, USA). RNA was confirmed pure by Nanodrop (Thermo Scientific, Waltham, MA, USA) measurements, quantified by Qubit RNA HS assay (Invitrogen), and checked for quality on a Lonza FlashGel RNA cassette gel (Lonza, Basel, Switzerland). Extracted RNA samples were stored at −80°C and mailed on dry-ice to the Joint Genome Institute for library preparation and sequencing.
Total RNA was treated with a TURBO DNA-free DNase treatment kit (Invitrogen, Waltham, MA, USA). RNA was confirmed pure by Nanodrop (Thermo Scientific, Waltham, MA, USA) measurements, quantified by Qubit RNA HS assay (Invitrogen), and checked for quality on a Lonza FlashGel RNA cassette gel (Lonza, Basel, Switzerland). Extracted RNA samples were stored at −80°C and mailed on dry-ice to the Joint Genome Institute for library preparation and sequencing.
7
Transcriptomic Analysis of Bile Stress
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For gene expression studies, RNA was extracted from mid-log phase cultures after 10-min exposure to 1% porcine bile or BHI (as a control) as previously described with minor modifications (Versantvoort et al., 2005 (link); Pleitner et al., 2014 (link)). Briefly, all experiments were conducted in three biological replicates on different days. For each strain and condition, 1ml of BHI or bile-exposed culture was collected and immediately added to ice-cold stop solution of 10% acid-phenol chloroform pH 4.5 (Invitrogen™, United States) in ethanol as outlined previously (Pleitner et al., 2014 (link)). DNase treatment was performed using TURBO DNA-free™ DNase treatment kit (Invitrogen, United States) according to the manufacturer’s protocol. Total RNA concentration was quantified using NanoDrop 2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA).
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