Total ribonucleic acid (RNA) was extracted from whole blood collected into PAXgene RNA Stabilization tubes and processed using a standard protocol (Qiagen, USA). The blood specimen was collected prior to administration of CTX. RNA concentration was measured by NanoDrop UV spectrophotometry (ThermoScientific, USA). RNA integrity was evaluated using the RNA 6000 Nano Assay (Agilent, USA). All RNA samples were determined to be of good quality (i.e., RNA Integrity Number (RIN) ≥ 8) and were retained for gene expression profiling.
Nanodrop uv spectrophotometry
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NanoDrop UV spectrophotometry is a compact and easy-to-use instrument designed for the quantification and analysis of nucleic acids and proteins. It utilizes a small sample volume (1-2 μL) to measure the absorbance of the sample, providing accurate results with minimal sample consumption.
Automatically generated - may contain errors
Lab products found in correlation
Rna 6000 nano assay, by Agilent Technologies (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Paxgene rna stabilization tubes, by Qiagen (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
Diastar rt kit, by Solgent (1 mentions)
Nuclease free water, by Merck Group (1 mentions)
Rocketscript reverse transcriptase, by Bioneer (1 mentions)
5 protocols using nanodrop uv spectrophotometry
1
RNA Extraction from Whole Blood
2
Isolation and Quality Assessment of Total RNA from Whole Blood
3
Transcriptome Analysis of Lemna minor
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Whole plants with fronds and roots were harvested, ground in liquid nitrogen, and total RNA was extracted from the samples using a RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) as per the manufacturer’s protocol. RNA concentrations were determined using a Nanodrop UV spectrophotometry (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was generated from 1 μg of total RNA from L. minor using a Diastar RT Kit (SolGent Co., Ltd, Daejeon, Korea).
Primers for quantitative reverse-transcription PCR (qRT-PCR) are summarized inTable 1 . qRT-PCR was performed using a CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, US). Each 10 μL reaction mixture contained 5 μL of 2X RT PCR Smart Mix (with SYBR Green) (SolGent Co., Ltd, Daejeon, Korea), 10 nM of each primer, and 1 μL of diluted first-strand cDNA. The cycling conditions were as follows: 95 °C for 5 min followed by 40 cycles of 94 °C for 30 s, 57 °C for 30 s, and 70 °C for 10 s in 96-well optical reaction plates. Cycle threshold (CT) values were determined for three biological replicates, with three technical replicates for each value. The expression levels of the reference gene (18S rRNA) and target gene (rbcL) tested were determined based on the Ct values and were calculated using the 2−△△CT method [44 (link)].
Primers for quantitative reverse-transcription PCR (qRT-PCR) are summarized in
4
RNA Extraction from Liver Samples
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RNA was extracted, DNaseI-treated, and purified from flash-frozen 50–100 mg liver samples following the standard procedures in the laboratory (Xu et al. 2013 (link); Xue et al. 2015 (link); Caballero-Solares et al. 2018 (link)). RNA integrity and purity were verified by 1% agarose gel electrophoresis and NanoDrop UV spectrophotometry (Thermo Fisher Scientific, Mississauga, ON, Canada), respectively. For all samples, no signs of RNA degradation were found (i.e., intact 28S and 18S ribosomal RNA bands), and A260/280 and A260/230 ratios were 2.1–2.3 and 1.9–2.4, respectively.
5
Droplet Digital PCR for RNA Quantification
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For ddPCR experiment, the sample mixtures were prepared in a 50 µL of master mix (one-step RocketScript™ Reverse Transcriptase, Bioneer, Korea) containing 1 µL of the template RNA, 1 µL of primers (10 µM each) and 1 µL of probes (10 µM) following the manufacturer’s protocol. Highly concentrated samples were diluted in nuclease-free water (Sigma-Aldrich, USA). The initial concentration was quantified by Nanodrop UV spectrophotometry (NanoDrop 2,000, Thermo Scientific, USA). The samples were partitioned by a droplet-based microfluidic chip as previously reported in our research group [26 ]. The droplets were collected in a tube and isothermal ddPCR was conducted at 39 °C for 20 min. The resulting droplets were pipetted onto slide glass and the fluorescence images were taken by a fluorescence microscopy (IX73, Olympus, Japan).
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