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Nanodrop nd 1000 instrument

Manufactured by Thermo Fisher Scientific
Sourced in United States

The NanoDrop ND-1000 is a spectrophotometer designed for the measurement of DNA, RNA, and protein samples. It utilizes a patented sample retention system that requires only 1-2 microliters of sample to perform measurements. The instrument is capable of determining the concentration and purity of nucleic acid and protein samples.

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30 protocols using nanodrop nd 1000 instrument

1

Microdissection and RNA Extraction from FFPE Tissues

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Manual microdissection was performed from each FFPE block to enrich for epithelial tissue before total RNA extraction as previously described.(21) As mentioned above, the target lesion was marked on the H&E slide for each case, which was then used to guide the removal of nontarget tissues (eg, normal parathyroid tissue) from unstained slides. After an overnight deparaffinization, the tissue area of interest was scraped off the slide into a microcentrifuge tube to facilitate total RNA extraction. MicroRNA extraction was performed by miRNeasy FFPE kit (QIAGEN Inc, Valencia, CA, USA), according to the manufacturer's instructions. The kit is optimized to isolate RNA molecules longer than 18 nucleotides from FFPE tissue samples, and to reverse as much formaldehyde modification as possible without further RNA degradation. Total RNA was extracted using the commercial RNeasy FFPE kit (QIAGEN GmbH, Hilden, Germany), according to the manufacturer's instructions.
The concentrations of the RNA samples were determined by OD260 using a NanoDrop ND‐1000 instrument (Thermo Fisher Scientific, Massachusetts, USA). The integrity of RNA was assessed by electrophoresis on a denaturing agarose gel.
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2

Quantifying Gene Expression in PBMC

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Total RNA was isolated from PBMCs using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at 4°C for 15 min according to the manufacturer's instructions. RNA concentrations were measured using a Nano-Drop ND-1000 instrument (Thermo Fisher Scientific, Inc.). The quality of RNA was determined via the optical density 260/280 ratio. RNA integrity was measured via 1% agarose gel electrophoresis. RT was performed to synthesize the cDNA with random primers in a ReverTraAca® RT-qPCR kit (Toyobo Life Science), which contained oligo-dT and random primers, according to the manufacturer's instructions. RT-qPCR was performed in triplicate using TB GreenTM Premix Ex Taq II (Takara Bio, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95°C for 3 min, followed by 40 cycles at 95°C for 12 sec, 62°C for 40 sec, and 72°C for 32 sec. The primer sequences are presented in Table SI. The expression level of each gene was normalized to the endogenous expression level of the β-actin transcript and was calculated using 2−∆∆Cq method (32 (link)). The data were analyzed using Applied Biosystems 7500 Manager software v2.3 (Thermo Fisher Scientific, Inc.).
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3

Circulating circRNA Expression in Ovarian Cancer

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The SBC Human (4 × 180 K) ceRNA array V1.0 (Shanghai Biotechnology Corporation, Shanghai, China) was used to analyze the expression of circRNA. RNAs were extracted from the plasma of four OC patients and four uterine myoma patients as controls by using Serum/Plasma Kit (QIAGEN, Dusseldorf, Germany). Then, NanoDrop ND-1000 instrument (Thermo, Waltham, MA, USA) and Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) were performed to detect the purity and integrity of the RNA. Total RNAs were amplified and labeled using Low Input Quick Amp Labeling Kit, One-Color (Agilent, Santa Clara, CA, USA) according to the manufacturer’s instructions and the labeled complementary RNAs (cRNAs) were purified by RNeasy Mini Kit (QIAGEN, GmbH, Germany). The labeled cRNAs were hybridized using Gene Expression Hybridization Kit (Agilent, Santa Clara, CA, USA). The completed hybridizations were scanned by an Agilent Microarray Scanner (Agilent, Santa Clara, CA, USA), and the Dye channel was set by the software: Green, Scan resolution = 3 μm, PMT 100%, 20 bit. Feature Extraction Software 10.7 (Agilent, Santa Clara, CA, USA) was used to read the data. Finally, quantile normalization and subsequent data processing were performed using the R software package. The screening threshold was set as fold change > 2.0 or < 0.5, P-value < 0.05.
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4

Somatic KRAS Mutations and MSI Analysis

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All 159 tumour samples were profiled for the presence of seven somatic KRAS mutations in codon 12 and 13 (12ALA, 12ASP, 12VAL, 12CYS, 12SER, 12ARG and 13ASP) using the Therascreen KRAS RGQ PCR kit (Qiagen) on formalin-fixed paraffin-embedded (FFPE) tumour tissue (analysis under ISO15189 accreditation). Other RAS mutations were not investigated. The MS status of all 159 tumour samples was analysed by fragment analysis on the ABI3130xl genetic analyser (Applied Biosystems) using the GeneMapper software 4 (Applied Biosystems). BAT25, BAT26, D2S123, D17S250, NR21, D18S55, NR24 and NR27 are the eight investigated repeat markers. Tumours were classified as MSI when instability of 25% of the markers was observed. The MSI patients could be divided into two groups, MSI-high and MSI-low, according to the number of aberrant markers. However, the numbers were too low for proper statistical analysis. In situations where the distinction was made, it is disclosed in the text.
DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer’s instructions. The quality and quantity of the DNA samples were measured with a NanoDrop ND-1000 instrument (Thermo Scientific).
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5

Quantification of miRNA and mRNA Expression in Mouse Liver

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Total RNA was extracted from mouse liver tissues or cells using Trizol reagent (15,596,026, Invitrogen, Carlsbad, CA), and the concentration and purity of total RNA were measured utilizing Nanodrop ND-1000 instrument (Thermo Fisher Scientific). For mRNA detection, complementary DNA (cDNA) was synthesized using the reverse transcription kit (RR047A, Takara, Japan). For miRNA detection, cDNA was obtained utilizing the miRNA First Strand cDNA Synthesis (Tailing Reaction) Kit (B532451-0010, Sangon, Shanghai, China). The expression of miR-223-3p was determined by RT-qPCR by means of TaqMan microRNA assays (Applied Biosystems) and ABI PRISM 7300 RT-PCR system (Applied Biosystems). Other genes were measured utilizing SYBR Green PCR Master Mix reagents (Applied Biosystems) and ABI PRISM 7900 Sequence Detection System (Applied Biosystems). Primers were synthesized by Takara (Table S2). The relative expression level was normalized to GAPDH or snRNA U6 expression and was calculated using the 2−ΔΔCt method.
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6

RNA-Seq Analysis of Plant Samples

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Total RNA was extracted from leaf samples using an RNA simple Total RNA kit (Tiangen, Beijing, China). The extracted RNA was quantified on a NanoDrop ND-1000 instrument (Thermo Scientific), and RNA integrity was determined by 1% agarose gel electrophoresis. Construction of RNA-Seq and cDNA libraries was carried out with 1 μg RNA per sample using a NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, United States). Sequencing of the eight generated libraries (R10, S10, R20, and S20, with two biological replicates per sample) was performed on an Illumina HiSeq 2000 system (Illumina, San Diego, CA, United States) by Biomarker Technologies Co. (Beijing, China).
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7

RNA-Seq analysis of ICSI blastocysts

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The blastocyst stage embryos were collected for sequencing. Five blastocysts were pooled for each treatment group (ICSI, ICSI-AOA and dICSI-AOA) in the same time from started injection and three independent replicates for each treatment procedure were collected. ICSI, ICSI-AOA and dICSI-AOA were named group A, B and D, respectively in the following sequencing. Total RNA was isolated from blastocyst of samples with TRIzol reagent (Life Technologies, Carlsbad, CA, USA) and GlycoBlue™ Coprecipitant (Ambion, AM9515) for facilitating good RNA recovery while increasing the size and visibility of the pellet. The RNA concentration of each sample was measured using the NanoDrop ND-1000 instrument (Thermo Fisher Scientific, Waltham, MA, USA). All RNA samples met standards of quality control according to the qualified ratio of OD260/OD280 ranged from 1.8 to 2.1. Transcriptome high-throughput sequencing was provided by CloudSeq Biotech Inc. (Shanghai, China). RNA preprocessing for constructing the sequencing library was implemented with the SMARTer Stranded Total RNA-Seq Kit (Takara). Libraries were controlled for quality and quantified using the BioAnalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA).
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8

Quantifying Gene Expression in Chicken Muscles

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Muscle samples were ground in liquid nitrogen, and about 30 mg of each sample was used to extract the total RNA with a TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The total RNA extracts were then treated with DNase I (Takara, Shiga, Japan) to eliminate the possible contamination of genomic DNA. The quantification and integrality of the RNA were checked with a NanoDrop ND-1000 instrument (Thermo Fisher Scientific, Wilmington, DE, USA) and formaldehyde-containing agarose electrophoresis, respectively. Two micrograms of the total RNA were subjected to reverse transcription to generate cDNA by using the GoScript Reverse Transcription System (Promega, Madison, WI, USA). A total of 2 μL of diluted cDNA (1:15) was subjected to real-time PCR using TB Green® Premix Ex Ta II (TaRaKa, Shiga, Japan). All the primers were designed by using Primer Premier 5 software (Premier Biosoft, Palo Alto, CA, USA), and the sequence of the primers was listed in Table 1. Chicken β-actin was selected as a reference gene.
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9

PGC-based DNA Nucleoside Desalting

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All samples described in the manuscript were desalted before being direct injected into the MS using a homemade filter composed of a plug of C18 filter pad (Empore) stuffed into a 200 μL pipet tip that was loaded with 60 μL (~1 μg) poly-graphitic carbon resin (PGC, HyperCarb, Thermo Scientific). The filter was washed with 100 μL 0.1% TFA prior to use. Digested DNA sample was mixed with 100 μL 0.1% TFA (pH = 2~3), loaded onto the filter and washed once with 100 μL 0.1% TFA. Nucleosides were eluted with 60 μL of a buffer composed as 60% acetonitrile (ACN) and 0.1% formic acid (FA) and dried in a vacuum centrifuge. For large amount of samples, commercially prepacked PGC 96 well plate is also available. The recovery from PGC nucleoside desalting was tested using extracted DNA from HepG2/C3A cells followed by digestion. The DNA concentration (A260 present in each preparation was assessed using a NanoDrop ND1000 instrument (Thermo Fisher) before and after desalting, confirming a recovery rate over 92%.
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10

Quantification of p53 Expression in Tissues

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Tissue samples in RNAlater (Sigma) were stored according to manufacturer’s instructions. Total RNA was extracted with TRIzol ™ Reagent (LifeTechnologies) per manufacturer’s protocol. Tissues (< 20 mg) were homogenized with 350 µl of TRIzol with TissueLyser (Qiagen). RNA concentration and quality were measured with NanoDrop ND-1000 instrument (Thermo Fisher Scientific). Reverse Transcriptase PCR was performed with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to manufacturer’s instructions. cDNA was synthesized from 250 ng of total RNA. Real time PCR was performed with SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad) in CFX96 Touch Real-Time PCR Detection System (Bio-Rad), using thermocycler conditions of 95 °C for 30 s, followed by 40 cycles of: 95 °C for 10 s, 60 °C for 30 s. All samples were evaluated in triplicate, with gene expression reported using the ∆CT method [46 (link)]. Wild type p53 expression (Fwd primer: GCGCCTATGGTTTCCATTTA, Rev primer: GGCAAAACAGCTTGTTGAGG) was compared to ACTB expression (Fwd primer: CAACCGTGAGAAGATGACTCAGA, Rev primer: CCCAGAGTCCATGACAATACCA) for each replicate [47 (link), 48 (link)].
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