Genomic DNA was extracted from the macrodissected FFPE tissues using Cobas DNA Sample Preparation Kit (Roche, Basil, Switzerland) according to manufacturer's protocol. Quantity and quality of isolated DNA was determined with NanoDrop 1000 UV Spectrophotometer (Thermo Scientific, Canton, GA, USA) and Qubit Fluorometer (ThermoFisher Scientific, Waltham, MA, USA). In a selected 22 samples, DNA from uterus or peripheral blood samples was isolated by using Cobas DNA Sample Preparation Kit (Roche, Basilea, Switzerland) or Blood Midi kit (A&A Biotechnology, Gdynia, Poland).
Nanodrop 1000 uv spectrophotometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NanoDrop 1000 is a UV spectrophotometer designed for the analysis of small volume samples. It allows for the measurement of nucleic acid and protein concentrations using only 1-2 microliters of sample.
Automatically generated - may contain errors
Lab products found in correlation
Easy nlc 1200, by Thermo Fisher Scientific (2 mentions)
Q exactive hf mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Cobas dna sample preparation kit, by Roche (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Hieff qpcr sybr green master mix, by Yeasen (1 mentions)
Primescript rt reagent kit with gdna eraser perfect real time, by Takara Bio (1 mentions)
Miseq pe250 platform, by Illumina (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Mobio powersoil dna isolation kit, by Qiagen (1 mentions)
Genomic tip 100 g, by Qiagen (1 mentions)
Protease k, by Qiagen (1 mentions)
Rnase a, by Nippon Gene (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Empore c18 disk, by 3M (1 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Ingenuity pathway analysis ipa, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
13 protocols using nanodrop 1000 uv spectrophotometer
1
FFPE DNA Extraction and Quantification
2
Quantifying Barley BAHD Gene Expression
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Total RNA was isolated from various barley tissues using total RNA Kit trnzol reagent (Tiangen, Beijing, China) following the manufacturer’s protocol. The quality and quantity of all the RNA samples were verified with a Nanodrop 1000 UV spectrophotometer (ThermoFisher Scientific, UT) and checked by 1% agarose gel electrophoresis. For qRT-PCR, first-strand cDNA was reverse transcribed from total RNA with the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time; TaKaRa). Real-time RT-PCR was performed with Hieff® qPCR SYBR Green Master Mix (Yeasen, Shanghai, China), using the real-time PCR system (Roche Light Cycler 96). The reaction procedure was a two-step method. The results of relative gene expression were calculated by 2ΔΔCt and the significance was analyzed by t-test66 (link). The HvACTIN (HORVU1Hr1G074350.1) was used as the internal reference gene67 (link). Primers used to quantify the expression of HvBAHDs in this study are listed (Supplementary Table S5 ).
3
Bacterial 16S rRNA Gene Sequencing
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The total genomic DNA of bacteria was extracted from the feces samples using the Ultra clean fecal DNA isolation kits (D1600, Solarbio Science & Technology, Beijing, China), according to the manufacturer’s protocol. The final DNA concentrations were determined by a Nano-Drop 1000 UV spectrophotometer (Thermo Scientific, Wilmington, DE, USA) at 260 and 280 nm, and the DNA quality was checked by 1% agarose gel electrophoresis.
The V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified for high-throughput sequencing by polymerase chain reactions (PCR) with the universal primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Purified amplicons were purified with the QIA quick PCR purification kits (28104, Qiagen, Valencia, CA, USA). The products were quantified using the QuantiFluorTM-ST fluorescent quantitation system (Promega, Madison, WI, USA), pooled in equimolar ratios and sequenced on an Illumina MiSeq PE250 platform (Illumina, San Diego, CA, USA) according to the standard protocols of Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). Alpha and beta diversity analyses were performed to describe the microbial diversity among samples using the online platform of the Majorbio Cloud Platform (www.majorbio.com , accessed on 20 December 2022).
The V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified for high-throughput sequencing by polymerase chain reactions (PCR) with the universal primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Purified amplicons were purified with the QIA quick PCR purification kits (28104, Qiagen, Valencia, CA, USA). The products were quantified using the QuantiFluorTM-ST fluorescent quantitation system (Promega, Madison, WI, USA), pooled in equimolar ratios and sequenced on an Illumina MiSeq PE250 platform (Illumina, San Diego, CA, USA) according to the standard protocols of Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). Alpha and beta diversity analyses were performed to describe the microbial diversity among samples using the online platform of the Majorbio Cloud Platform (
4
Soil DNA Extraction and Quantification
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MoBio Powersoil® DNA Isolation Kit (MoBio Laboratories, USA) was used to extract the total genomic DNA from each soil sample. The DNA quality was determined by gel electrophoresis (1% agarose). The DNA concentration was determined by NanoDrop 1000 UV spectrophotometer (Thermo Scientific, USA).
5
Maize Genomic DNA Extraction and Quantification
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Non-GM maize kernels (50 g) were ground
using a food processor. Non-GM or GM maize genomic DNA (gDNA) was
extracted and purified from 1 g of corn powder using an anion-exchange
resin Genomic-tip 100/G (QIAGEN, Hilden, Germany). The powder was
completely resuspended with 7 mL of buffer G2, 100 μL of protease
K (QIAGEN), and 5 μL of RNase A (100 mg/mL) (NIPPON GENE) at
50 °C for 60 min. The supernatant was loaded onto Genomic-tip
equilibrated with 4 mL buffer QBT. The Genomic-tip was washed twice
with 7.5 mL buffer QC. Maize gDNA was eluted with 3 mL pre-warmed
(50 °C) buffer QF and purified by isopropanol precipitation.
The obtained DNA pellet was rinsed with 70% ethanol, dried, and resuspended
with ultrapure water. The quantity and quality of DNA were evaluated
using a NanoDrop1000 UV spectrophotometer (Thermo Fisher Scientific,
Waltham, MA). Each sample was diluted to a concentration of 10 ng/μL
in ultrapure water. MON863 standard samples were prepared by diluting
GM maize gDNA with non-GM maize gDNA to concentrations of 0.2, 0.1,
0.05, 0.02, 0.01, 0.005, and 0.001%. MON863 standard samples are hereafter
defined as MON863-RM.
using a food processor. Non-GM or GM maize genomic DNA (gDNA) was
extracted and purified from 1 g of corn powder using an anion-exchange
resin Genomic-tip 100/G (QIAGEN, Hilden, Germany). The powder was
completely resuspended with 7 mL of buffer G2, 100 μL of protease
K (QIAGEN), and 5 μL of RNase A (100 mg/mL) (NIPPON GENE) at
50 °C for 60 min. The supernatant was loaded onto Genomic-tip
equilibrated with 4 mL buffer QBT. The Genomic-tip was washed twice
with 7.5 mL buffer QC. Maize gDNA was eluted with 3 mL pre-warmed
(50 °C) buffer QF and purified by isopropanol precipitation.
The obtained DNA pellet was rinsed with 70% ethanol, dried, and resuspended
with ultrapure water. The quantity and quality of DNA were evaluated
using a NanoDrop1000 UV spectrophotometer (Thermo Fisher Scientific,
Waltham, MA). Each sample was diluted to a concentration of 10 ng/μL
in ultrapure water. MON863 standard samples were prepared by diluting
GM maize gDNA with non-GM maize gDNA to concentrations of 0.2, 0.1,
0.05, 0.02, 0.01, 0.005, and 0.001%. MON863 standard samples are hereafter
defined as MON863-RM.
6
Serum RNA Extraction and cDNA Synthesis
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Total RNA was extracted from 250 µl serum using the trizol LS reagent (Invitrogen, USA) according to the manufacturer’s instructions. RNA concentration and purity were determined using a NanoDrop 1000 UV spectrophotometer (Thermo, Wilmington, DE, USA). Reverse transcription of cDNA can be performed only when each sample contained 3000 ng RNA and the optical density ratio (260/280) is between 1.8 and 2.0. The EvoScript Universal cDNA Master reverse transcription kit (Roche, Basel, Switzerland) was used to obtain cDNA. The reaction conditions for the synthesis of cDNA were as follows: 15 min at 42℃, 5 min at 85℃, 15 min at 65℃ and 5 min at 4℃. The obtained cDNA was stored at − 80 °C for future use.
7
Quantitative Proteomics Analysis Protocol
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The immuno-precipitates were denatured with 8 M urea and cysteine residues reduced with dithiothreitol then alkylated with iodoacetamide. After digestion with trypsin (37 0C, 16 h), the samples were desalted using Empore C18 disks (3M, Cat# 2215). Based on using UV absorption measurements with a NanoDrop-1000 UV spectrophotometer (Thermo Scientific), equivalent aliquots of the digested peptides were analyzed in triplicate with an Easy-nLC 1200 coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). A 20 × 0.1 mm i.d. pre-column was used in conjunction with a 150 mm × 75 μm i.d. analytical column, both packed with 5 μm Reprosil C18-bonded silica (Dr Maisch GmbH). A separation gradient from 8 to 45% B in 78 min was used at a flow rate of 300 nl/min. The mobile phase compositions were, water with 0.1 % formic acid (A) and 80% acetonitrile 0.1% formic acid (B). The MS/MS data were acquired in positive ion mode with a data dependent acquisition setting for higher-energy C-trap dissociation (HCD) of the 10 most intense ions (m/z 300-2000, charge states > 1+). Further details of the preparation and instrument parameters are available as supplementary information. The LC-MS/MS discovery data are available via the PRIDE (Vizcaíno et al., 2016 (link)) partner repository from the ProteomeXchange Consortium with the dataset identifier PXD008983.
8
Endometrial Cancer Tissue Procurement
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A total of 18 available and suitable tissues were selected from patients with G3 EEC and with at least 2 years of follow up (up to 1 February 2018). All patients underwent surgical treatment between May 2013 and November 2015, at the Obstetrics and Gynecology Unit, Careggi University Hospital (Florence, Italy). Patients were treated depending on International Federation of Gynecology and Obstetrics (FIGO staging), grading and European Society of Gynecological Oncology (ESGO) risk (52 (link)). Tumor samples were collected at the time of surgery, following acquisition written informed consent from the patients. The study was approved by the Ethics Committee of Careggi University Hospital. Patients' demographic, clinical and pathological features are presented in Table I .
Tumor samples were immediately fixed in formalin and processed for paraffin embedding. The resulting formalin-fixed paraffin-embedded (FFPE) tissues were examined histologically by pathologists at Careggi University Hospital for the selection of areas containing >70% of tumor cells.
DNA was extracted from slices using a QIAamp DNA FFPE tissue kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's protocol. DNA quality (ratio R) and quantity (DNA concentration) were determined using a spectrophotometer (NanoDrop® 1000 UV spectrophotometer; Thermo Fisher Scientific, Inc., Waltham, MA, USA).
Tumor samples were immediately fixed in formalin and processed for paraffin embedding. The resulting formalin-fixed paraffin-embedded (FFPE) tissues were examined histologically by pathologists at Careggi University Hospital for the selection of areas containing >70% of tumor cells.
DNA was extracted from slices using a QIAamp DNA FFPE tissue kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's protocol. DNA quality (ratio R) and quantity (DNA concentration) were determined using a spectrophotometer (NanoDrop® 1000 UV spectrophotometer; Thermo Fisher Scientific, Inc., Waltham, MA, USA).
9
Transcriptome Analysis of Naive and Memory CD4+ T Cells
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RNA was extracted from naïve and memory CD4+ T-cells at indicated experimental time-points using RNeasy Mini kit (Qiagen), as per manufacturer's instructions. RNA was quantified using a Nanodrop 1,000 UV Spectrophotometer (Thermo Fisher Scientific) prior to generation/hybridization of cRNA to the Illumina Human HT-12 v4.0 microarray (Illumina, San Diego, USA). Raw data processing and further data analysis was performed in R software version 3.3.0, using the Bioconductor packages. Variance-stabilizing transformation (VST) and Robust Spline Normalization (RSN) were carried out in the lumi package and differentially expressed genes were identified using the moderated paired t-test implemented in the limma package, with a fold-change of 1.5 and p < 0.05 after correction for multiple testing using the Benjamini-Hochberg method. Pathway analysis of differentially expressed gene lists was carried out using Ingenuity Pathway Analysis (IPA; Qiagen). Hypergeometric testing was carried out in order to determine the probability of genes differentially expressed between early RA and disease controls occurring in IL-6 exposed naïve CD4+ T cells. Expression data used in this study are available in the Gene Expression Omnibus database (accession number GSE131866 ; https://www.ncbi.nlm.nih.gov/geo ).
10
Quantifying Lens Capsule RNA Expression
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Total RNAs of the lens capsule samples of Hypothermia Group (n = 3) and the Nomal Control Group (n = 3) were extracted using Trizol Reagent (Thermo Fisher), four corneas served as one biological replicate. RNA concentrations and OD260/OD280 ratios (1.9–2.0) were determined using a Nanodrop 1,000 UV spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). Approximately 1,500 ng RNA was used per reverse transcription reaction (PrimeScriptRT Reagent Kit; TaKaRa, Shiga, Japan). The TB Green Premix Ex Taq Kit (TaKaRa) was used for quantitative real-time PCR on a 7500 Fast Real-Time PCR System (ABI, Loma Linda, CA, United States), with a 2-step method. The expression levels of the target genes were normalized to that of β-actin and were calculated using the 2–ΔΔCT method and indicated as means ± SE, statistical analysis was conducted using t-test. The data are presented as fold-changes. The primers (TSINGKE, Beijing, China) are shown in Supplementary Table 1 .
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