A drop of non-preserved tetracaine was firstly instilled into the conjunctival fornix. After the stinging sensation had resolved, a sterile cotton swab was used to collect the microbes from the lower conjunctival fornix using a gentle rolling action (up to eight strokes). The procedure was then repeated for the opposite eye. The cotton swabs from both eyes were combined and then soaked in 650 μl of DNA/RNA Shield (Zymo Research Corp., Irvine, CA) reagent, immediately homogenized for 30 s, transferred to ice for 1 min, and further homogenized for another 30 s. Homogenized samples were stored at 4°C until further processing (within 1 week). Total DNA was extracted with ZR-Duet DNA/RNA MiniPrep (Zymo Research, Irvine, CA). Empty swabs following the same procedure were used as control. An empty swab was an unused swab that was opened under the same room and conditions as the participants and then homogenized and processed as if it has been used on a participant.
Zr duet dna rna miniprep
Manufactured by Zymo Research
Sourced in United States
The ZR-Duet DNA/RNA MiniPrep is a lab equipment product designed for the simultaneous extraction of both DNA and RNA from a single sample. It provides a convenient and efficient method for the purification of nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Dna rna shield, by Zymo Research (1 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
7500 fast, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Infinium humanmethylation450 beadchip, by Illumina (1 mentions)
Hiseq 4000 system, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Abi 7300 instrument, by Thermo Fisher Scientific (1 mentions)
Revertra ace qpcr rt master mix with gdna remover kit, by Toyobo (1 mentions)
Kapa sybr fast qpcr kit, by Roche (1 mentions)
Triad multi mode microplate reader, by Dynex (1 mentions)
Methylated dna quantification kit, by Abcam (1 mentions)
Rnase free dnase 1, by Thermo Fisher Scientific (1 mentions)
Maxima first strand cdna synthesis kit for rt qpcr, by Thermo Fisher Scientific (1 mentions)
Methylation gold kit, by Zymo Research (1 mentions)
Qiashredder, by Qiagen (1 mentions)
13 protocols using zr duet dna rna miniprep
1
Conjunctival Microbiome Sampling Protocol
2
Gill Tissue Sampling and RNA Extraction
3
Quantitative Real-Time RT-PCR Analysis
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Total RNA was extracted using ZR-Duet DNA/RNA MiniPrep (Zymo Research), according to the manufacturer's guidelines. RNA from all samples was treated with DNase I (Sigma-Aldrich) to remove any genomic DNA contamination. The purified RNA was quantified using NanoDrop TM 1,000 Spectrophotometer (Thermo Scientific) and 50-200 ng of RNA were used to synthesize complementary DNA (cDNA) using oligo (dT) 20 or random primers (300 ng/µL; Invitrogen) and Superscript III reverse transcriptase (Invitrogen). mRNA levels were determined by Real-Time RT-PCR using the appropriate set of primers (Table 1) and Power SYBR Green PCR Master mix (Applied Biosystems) on a 7500 Fast or the ViiA TM 7 Real-Time PCR System (both from Applied Biosystems). Relative mRNA levels, normalized to β-actin, were calculated using the 2-CT or 2-CT method.
4
Genome-wide DNA Methylation Profiling
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Chromosomal DNA from cells grown in 25 cm2 culture flasks was isolated using a ZR-Duet DNA/RNA MiniPrep (Zymo Research, Irvine, CA) with a final elution in 40 μl of distilled water. A quantity of 50 ng of chromosomal DNA was treated with bisulfite and applied to the Infinium HumanMethylation450 Beadchip to monitor genome-wide methylation (> 485,000 CpG sites) (Illumina, San Diego, CA). The methylation level was presented as a methylation index (β) as described previously 21 (link). Briefly, β values ranging from 0 (no methylation) to 1 (100% methylation) were calculated; the higher the value, the greater the level of methylation in proton beam-treated samples relative to non-treated cells. The results of the array have been deposited on the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo /) and are accessible with the accession number GSE70024.
5
PDX Tumor Transcriptome Analysis
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Tumor tissues from treated and control PDX lines were harvested when the tumor volume in the control group exceeded 1500 mm3. After the tumor-bearing mice were sacrificed, the tumors were cut into 4-µm thick pieces that were immediately frozen in liquid nitrogen. The frozen tissue was crushed using a Cryo-Press (Microtec Co., Ltd.). Total RNA was isolated and purified with ZR-Duet DNA/RNA MiniPrep (Zymo Research Corp.). RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.), followed by next-generation sequencing (NGS) on an Illumina HiSeq 4000 system at BGI with paired-end sequencing. The read lengths were 2×100 bp for GEM monotherapy and 2×150 bp for GEM + nab-PTX combination therapy.
6
Exosome Uptake by Dermal Fibroblasts
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NHDF fibroblasts were cultured in a 6-well plate (1.2 × 106 cells/well) and grown in a dFBS medium. Cells were treated with the obtained exosome isolates (100 ng) for 24 h. Total RNA was then isolated from each sample using the ZR-Duet™ DNA/RNA MiniPrep (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. Controls consisted of untreated NHDF cells. The samples were prepared in triplicate.
7
Quantitative RNA Expression Analysis of Cultured Cells and Tissues
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Total RNA from cultured cells in 75-cm2 and approximately 200 mg of tissues was isolated using the ZR-DuetDNA/RNA MiniPrep (Zymo Research, Irvine, CA, USA) with a final elution in 45 μl of distilled water. Reverse transcription was conducted using 2 μg of total RNA with a ReverTra Ace qPCR RT MasterMix with gDNA Remover kit (Toyobo, Osaka, Japan). Gene expression was measured using a Kapa SYBR Fast qPCR Kit (Kapa Biosystems, Wilmington, MA, USA) on an ABI 7300 instrument (Applied Biosystems, Foster City, CA, USA). Each of the samples was performed in triplicate. GAPDH was used for normalization of each gene expression. The 2−ΔCt method was used for the quantifying process, where ΔCt = (CtTARGET − CtGAPDH). Primer sequences used for PCR are shown in the Supplementary Table S1 .
8
Quantifying Global DNA Methylation
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Total DNA was prepared using the ZR-Duet™ DNA/ RNA MiniPrep (Zymo Research) according to the manufacturer's protocol. For the measurement of global DNA methylation status, Methylated DNA Quantification Kit was used (Abcam). To quantify the absolute amount of methylated DNA a standard curve was generated (0.5, 1.0, 2.0, 5.0 and 10.0 ng/µl for 5-mC), and the OD (optical density) values versus the amount of positive control at each concentration point were plotted. The slope (OD/ng) of the standard curve was determined using linear regression. The amount (ng) of methylated DNA (5-mC) was calculated by subtracting the negative control OD from sample OD and divided by the slope (OD/ng) and multiplied by 2, which is a factor to normalize 5-mC in the positive control to 100%, as the positive control contains only 50% of 5-mC, according to the manufacturer's protocol. The percentage of methylated DNA was calculated by dividing the amount of 5-mC (ng) by the amount of input sample DNA (ng) and multiplying by 100%. Absorbance at 450 nm was measured using with ELISA plate reader (Dynex Technologies Triad Multi-Mode Microplate Reader). Samples were prepared in triplicate.
9
RNA Isolation and Reverse Transcription
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After 48 h of incubation under the conditions above mentioned, total RNA was isolated from tumor cells (A-549, A-427, MCF-7) and fibroblasts (MRC-5) using ZR-Duet DNA/RNA Miniprep according to manufacturer's instructions (Zymo Research, Irvine, CA, USA). Total RNA was treated with RNAse free DNAse I (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's protocol. Quality and quantity of RNA were evaluated by A260 and A280 using NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). Total RNA was reverse-transcribed using the kit Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific, Waltham, MA, USA). The cDNA obtained was stored at−20°C for further analysis.
10
Keratinocyte Stimulation by dsRNA and dsDNA
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Human adult epidermal keratinocytes (HEKa, Life technologies, Carlsbad, USA) were maintained in medium 154 with human keratinocyte growth supplement (HKGS) (Life technologies) and cultured at 37 °C with 5% CO2. The day before stimulation, cells between passage 3 to 6 were plated in 6-well plates at 2 × 105 cells/well in 2 ml of complete growth medium. At the time of stimulation, cells were treated with 1 μg/ml double-stranded RNA (dsRNA) analog poly(I:C) (Polyinosinic acid: Polycytidylic acid) or 1 μg/ml double-stranded DNA (dsDNA) analog poly(dA:dT) (poly(deoxyadenylic-deoxythymidylic) acid sodium salt) (Invivogen, San Diego, CA, USA). Experiments were performed in duplicate and repeated in two independent experiments. Cells were collected 24 hours after stimulation and RNA/DNA co-isolated using ZR-Duet™ DNA/RNA MiniPrep from Zymo Research, according to the manufacturer’s manual (Irvine, CA, USA).
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