Total RNA was extracted with a Picopure RNA isolation kit (Molecular Devices, Sunnyvale, CA) using DNase I. Quantification of total RNA was determined by the fluorescence based method, using a RiboGreen RNA Quantification kit (Molecular Probes, Eugene, OR). The integrity of RNA from aleurone cells and starchy endosperm was assessed using a 2100 Bioanalyzer (Agilent technologies, Santa Clara, CA). The average RNA integrity number (RIN) with clear rRNA peaks from each tissue exceeded 7.0 for the three biological replications (Additional file 1 : Table S1).
Ribogreen rna quantification kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The RiboGreen RNA quantification kit is a fluorescent dye-based assay designed to accurately measure the concentration of RNA in a sample. The kit provides a sensitive and rapid method for quantifying RNA, making it a useful tool for various applications in molecular biology and life science research.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Mirneasy kit, by Qiagen (2 mentions)
Rnalater, by Thermo Fisher Scientific (2 mentions)
Truseq stranded mrna library prep kit, by Illumina (2 mentions)
Bioanalyzer tapestation, by Illumina (2 mentions)
Truseq stranded mrna libraries with unique dual indexed, by Illumina (2 mentions)
Tapestation, by Agilent Technologies (2 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Picopure rna isolation kit, by Molecular Devices (1 mentions)
Rnase inhibitor, by Roche (1 mentions)
Nanodrop system, by Thermo Fisher Scientific (1 mentions)
Amv reverse transcriptase, by Promega (1 mentions)
Rnase free dnase set, by Qiagen (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (1 mentions)
Mmessage mmachine t7 ultra transcription kit, by Thermo Fisher Scientific (1 mentions)
Paxgene rna tube, by Qiagen (1 mentions)
V4 chemistry, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Fluostar optima plate reader, by BMG Labtech (1 mentions)
14 protocols using ribogreen rna quantification kit
1
High-Quality RNA Extraction from Plant Tissues
2
RNA Extraction and cDNA Synthesis
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RASF were harvested and total RNA extracted (RNeasy Mini Kit, Qiagen, Germany). Remaining DNA was removed using the RNase-free DNase Set (Qiagen). RNA concentrations were quantified (Ribogreen RNA quantification kit, Molecular Probes, Netherlands, or Nanodrop system, Thermo Fisher) and RNA stored at − 80 °C.
cDNA was synthesized using 150 ng RNA, 5 mM Tris-HCl (pH 8.3, 25 °C), 50 mM KCl, 1 mM MgCl2, 0.5 mM spermidine, 1 mM dithiothreitol, 1 mM each dNTP (Roche, Germany), A260 unit random primer (Roche), 1.6 U/μl RNase inhibitor (Roche), and 1.3 U/μl AMV reverse transcriptase (Promega, Germany). Conditions were 25 °C 10 min, 42 °C 60 min, and 99 °C 5 min. cDNA was stored at − 20 °C.
cDNA was synthesized using 150 ng RNA, 5 mM Tris-HCl (pH 8.3, 25 °C), 50 mM KCl, 1 mM MgCl2, 0.5 mM spermidine, 1 mM dithiothreitol, 1 mM each dNTP (Roche, Germany), A260 unit random primer (Roche), 1.6 U/μl RNase inhibitor (Roche), and 1.3 U/μl AMV reverse transcriptase (Promega, Germany). Conditions were 25 °C 10 min, 42 °C 60 min, and 99 °C 5 min. cDNA was stored at − 20 °C.
3
RNA Extraction from Cell Pellets
4
Quantification of ZIKV RNA Levels
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Total RNA was extracted using PureLink RNA Mini Kit (Invitrogen) combined with TRIZOL purification and treated with Turbo DNA-free Kit (Invitrogen). ZIKV RNA copies were quantified by quantitative reverse transcriptase-PCR (qRT-PCR). Primers and probe sets were designed according to a previously reported ZIKV1162c set.84 (link) A standard RNA covering the probe-target was synthesized from a DNA template by mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Ambion) and quantified by RiboGreen RNA Quantification Kit (Invitrogen). For samples in which no qRT-PCR signal was detected, zero values were replaced with half the minimum experimentally observed value to allow log transformations. ZIKV copy number for each brain was determined relative to a standard curve from 10-fold serial dilutions of a ZIKV RNA standard. RNA copy number was calculated as previously described.85 (link) For downstream morphometric and immunohistochemical analysis, we divided the groups into ZIKV-injected and Mock-injected and did not take into account the results of ZIKV qRT-PCR to avoid introducing biases. For and transcriptomic and proteomic analyses, Mock-injected control embryos unambiguously negative for ZIKV qRT-PCR were selected.
5
RNA-Seq Analysis of Sigmoid Colon Biopsies
6
RNA Extraction and Sequencing from Whole Blood
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Whole blood was drawn from patients directly into PAXgene RNA tubes (QIAGEN Inc.). These tubes were frozen and shipped to the University of Minnesota. We used the “whole blood PAXgene Blood RNA kit” to extract the ribonucleic acid (RNA) from blood samples (QIAGEN Inc.) according to the manufacturer’s protocol. One microgram of total RNA was measured by RiboGreen RNA Quantification kit (Invitrogen Inc. USA). One microgram was submitted to the University of Minnesota Biomedical Genomics facility for quality controls, assessed by Agilent 2100Bioanalyzer (Agilent Technologies Inc. USA). The Clontech StrandedRNA Pico Mammalian kit was used for library creation. Paired-end (2 × 125 bp or x50bp) sequencing was done on a HiSeq2500 instrument, for 125 cycles, using v4 chemistry (Illumina Inc. USA). The fastq files from this study are deposited in the National Center for Biotechnology Information’s Gene Expression Omnibus and are accessible through GEO Series, ticket number is GSE162914.
7
Saliva RNA Extraction and Quantification
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Cell-free saliva, salivary ELMs, and ELM-depleted saliva supernatant were treated with RNase cocktail (final concentration 100 U/ml) with or without 1% Triton X-100 (Tx) at room temperature for 20 min. RNA was extracted from these processed samples using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. The isolated RNA was quantified using the RiboGreen RNA quantification Kit (Invitrogen) and analyzed by reverse transcription PCR (RT-PCR) followed by quantitative PCR (qPCR).
8
RNA Extraction and Reverse Transcription
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3–5 mm3 of frozen tissue from the left midfrontal region and temporal lobe was homogenised in TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in a Precellys 24 homogenizer, incubated for 3 min in chloroform and centrifuged at 12,000g for 15 min at 4 °C. The upper aqueous phase was separated and mixed with an equal volume of isopropanol and 30 µg of glycogen (Sigma-Aldrich), incubated for 10 min, and centrifuged at 12,000g for 10 min at 4 °C for ribonucleic acid (RNA) precipitation. The RNA pellet was washed with 75 % ethanol, re-suspended in water (Sigma-Aldrich), and treated with DNase-I (40 U, Roche Diagnostics Ltd., West Sussex, UK) to remove genomic DNA. RNA concentration was measured using the Ribogreen RNA Quantification Kit (Invitrogen) and a FluoStar OPTIMA plate reader (BMG Labtech). RNA was reverse transcribed to complementary DNA (cDNA) using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA): 100 ng of RNA in a total volume of 100 µL was incubated at 25 °C for 10 min, 37 °C for 2 h, followed by inactivation at 85 °C for 5 s. cDNA concentration was determined using the Picogreen DNA quantification kit (Invitrogen) and FluoStar OPTIMA plate reader (BMG Labtech) according to the manufacturer’s instructions.
9
RNA Extraction and CsTFA Separation
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RNA was extracted from 2 ml of each sludge sample from the 13C and unlabeled treatments done in triplicate after an 8-h incubation. Total nucleic acids extraction was performed using a direct lysis protocol involving bead beating [44 (link)]. Then, RNA was purified by DNA digestion with a DNase (RQ1; Promega, Japan). Total RNA was quantified using a RiboGreen RNA quantification kit (Invitrogen, CA, USA) and a microplate reader (SH-900Lab; Corona, Japan). Five-hundred nanograms of RNA mixed with cesium trifluoroacetate (CsTFA) solution (Wako) was subjected in triplicate to ultra-centrifugation with 128,000 g for >60 h at 20 °C [45 (link)]. Gradients of density-separated RNAs were fractionated, and the CsTFA buoyant density (BD) of each fraction was determined with a refractometer (AR200; Reichert, NY, USA) [45 (link)].
10
Density Gradient Analysis of Stable Isotope-Labeled RNA
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RNA was extracted from 0.5 ml of slurry samples that were obtained after 2- and 6-h incubations with 13C-labeled and unlabeled dead E. coli cells using a direct lysis protocol involving bead beating (Noll et al., 2005 (link)). Total RNA was quantified using the Ribogreen RNA quantification kit (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions. RNA extracts (500 ng RNA) from 13C-labeled and unlabeled samples were mixed with cesium trifluoroacetate (CsTFA) (Wako Pure Chemical Industries Ltd., Osaka, Japan) solution. The mixture was subjected to equilibrium density gradient centrifugation as previously described (Lueders et al., 2004 (link)). The RNA density gradients were fractionated, and their CsTFA buoyant density (BD) was determined (Lueders et al., 2004 (link)).
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