Total RNA was extracted from bone cores using TRIzol according to the manufacturer’s instructions. The pellet was then resuspended in RNase free water and measurement of RNA yield was performed using a NanoDrop 1000A Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription reaction was performed for cDNA synthesis using High Capacity cDNA RT kit (Applied Biosystems, Foster City, CA 94404) according to the manufacturer’s instructions. Samples were then stored at −80°C. Quantitative real-time PCR (qPCR) amplification was performed using QuaniTect® Probe PCR kit (QIAGEN Inc, Helden Germany). Taqman assays were used to quantify expression of genes listed in Table 4 . Sequence-specific probes are listed in Table 4 . The housekeeping gene GAPDH was analyzed in every sample and used as an endogenous control for data normalization. Samples were assayed in triplicate. Relative quantification studies of threshold cycle (Ct) were performed with Sequence Detector software (Applied Biosystems, Foster City, CA). Relative gene expression in patient samples was expressed relative to the healthy controls.
Nanodrop 1000a spectrophotometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nanodrop 1000A spectrophotometer is a compact, microvolume UV-Vis spectrophotometer designed for the quantification and analysis of nucleic acids and proteins. It features a patented sample retention technology that allows for accurate measurements of small sample volumes (0.5-2 μL) without the need for cuvettes or dilutions.
Automatically generated - may contain errors
Lab products found in correlation
Sequence detector software, by Thermo Fisher Scientific (3 mentions)
High capacity cdna rt kit, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Quanitect probe pcr kit, by Qiagen (2 mentions)
Rnease kit, by Qiagen (2 mentions)
Humanht 12 v4, by Illumina (2 mentions)
Bioanalyzer rna nano chip, by Agilent Technologies (2 mentions)
Quantitect probe pcr kit, by Qiagen (1 mentions)
Rneasy, by Qiagen (1 mentions)
Totalprep rna amplification kit, by Thermo Fisher Scientific (1 mentions)
Totalprep 96 rna amplification kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Open array real time pcr platform, by Thermo Fisher Scientific (1 mentions)
Ambion ribopure kit, by Thermo Fisher Scientific (1 mentions)
Quantstudio 12k flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Rnase free water, by Thermo Fisher Scientific (1 mentions)
Mirvana paris kit, by Thermo Fisher Scientific (1 mentions)
Masterpure rna purification kit, by Illumina (1 mentions)
Pre mir, by Thermo Fisher Scientific (1 mentions)
13 protocols using nanodrop 1000a spectrophotometer
1
Bone RNA Extraction and qPCR Protocol
2
Venous Blood DNA Extraction Protocol
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Whole venous blood specimens were gathered on Vacutainer ethylenediaminetetraacetic acid (EDTA) blood collection tubes from all patients. Genomic DNA extraction by column-based extraction kits (QIAamp DNA Blood Mini Kit, Catalogue number: 51104) was performed depending on the manufacturer’s manuals. The purity and concentration of DNA were identified by the Thermo Scientific NanoDrop™ 1000A Spectrophotometer at 260 and 280 nm.
3
Bone RNA Extraction and qPCR Protocol
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Total RNA was extracted from bone cores using TRIzol according to the manufacturer’s instructions. The pellet was then resuspended in RNase free water and measurement of RNA yield was performed using a NanoDrop 1000A Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription reaction was performed for cDNA synthesis using High Capacity cDNA RT kit (Applied Biosystems, Foster City, CA 94404) according to the manufacturer’s instructions. Samples were then stored at −80°C. Quantitative real-time PCR (qPCR) amplification was performed using QuaniTect® Probe PCR kit (QIAGEN Inc, Helden Germany). Taqman assays were used to quantify expression of genes listed in Table 4 . Sequence-specific probes are listed in Table 4 . The housekeeping gene GAPDH was analyzed in every sample and used as an endogenous control for data normalization. Samples were assayed in triplicate. Relative quantification studies of threshold cycle (Ct) were performed with Sequence Detector software (Applied Biosystems, Foster City, CA). Relative gene expression in patient samples was expressed relative to the healthy controls.
4
Quantitative Analysis of Osteoblast Gene Expression
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Total RNA was extracted from confluent osteoblasts using RNeasy (Qiagen) according to the manufacturer's instructions. Measurement of RNA yield was performed using a NanoDrop 1000A Spectrophotometer (Thermo Fisher Scientific). A reverse transcription reaction was performed for cDNA synthesis using High Capacity cDNA RT kit (Applied Biosystems) according to the manufacturer's instructions. Quantitative real-time PCR (qPCR) amplification was performed using QuantiTect ® Probe PCR kit (Qiagen) to assess adipsin/complement factor D (CFD) (Hs00157263_m1), CCAAT/enhancer-binding protein alpha (CEBPA) (Hs00269972_S1), lipoprotein lipase (LPL) (Hs00173425_m1), runt-related transcription factor 1 (RUNX) (Hs00231692_m1), vitamin D receptor (VDR) (Hs01045840_m1), 25-hydroxyvitamin D3 1-alpha-hydroxylase (CYP27B1) (Hs00168017_m1), cytochrome P450 family 24 subfamily A member 1 (CYP24A1) (Hs00167999_m1) expression. The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control for data normalization. Samples were assayed in triplicate for these analyses and the coefficient of variation between technical replicates was less than or equal to 2%. Relative quantification studies of threshold cycle (Ct) were performed with Sequence Detector software (Applied Biosystems).
5
Biotin-labeled RNA Analysis of bLf Effects
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HT-29 cells (1.6 x 10 4 /cm 2 , 6-well plate) were grown over night and then treated with bLf (800 µg/mL), LfcinB-C (400 µg/mL), or LfcinB-L (400 µg/mL) for 12 h. RNA was extracted from the harvested cells using Trizol Reagent (Invitrogen). RNA was then purified by an RNease kit (Qiagen) according to the manufacturer's instructions. Measurement of RNA yield was performed using a NanoDrop 1000A Spectrophotometer (Thermo Fisher Scientific, Waltham, MA), and RNA quality was verified by using Bioanalyzer RNA Nano Chips (Agilent Technologies, Inc., Santa Clara, CA) following the manufacturer's procedure.
Total RNA samples were amplified and labeled with biotinylated nucleotides using a kit (Ambion, TotalPrep RNA Amplification Kit). Bioanalyzer analysis was then performed to verify if cRNA was at the expected 1.2 kb average size before applying to bead chips (HumanHT-12 v.4.0, Illumina, San Diego, CA). Bead chips were scanned with the Illumina iScan using standard conditions.
Total RNA samples were amplified and labeled with biotinylated nucleotides using a kit (Ambion, TotalPrep RNA Amplification Kit). Bioanalyzer analysis was then performed to verify if cRNA was at the expected 1.2 kb average size before applying to bead chips (HumanHT-12 v.4.0, Illumina, San Diego, CA). Bead chips were scanned with the Illumina iScan using standard conditions.
6
Optimization of Gene Expression Analysis
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HDFn (6-well plate, 2.5 x 10 3 /cm 2 ) were grown for 24 h and then treated with CbLf (100 µg/mL), SLs (10 µg/mL), or CbLf-SLs-assembly for 24 h. RNA was subsequently extracted with Trizol (Invitrogen), and then purified by the RNease kit (Qiagen) according to the manufacturer's instructions. Measurement of RNA yield was performed using a NanoDrop 1000A Spectrophotometer (Thermo Fisher Scientific, Waltham, MA), and RNA quality was evaluated by Bioanalyzer RNA Nano Chips (Agilent Technologies, Inc., Santa Clara, CA) following the manufacturer's procedure.
Total RNA samples were amplified and labeled with biotinylated nucleotides using a kit (Ambion, TotalPrep -96 RNA Amplification Kit). Bioanalyzer analysis was then carried out to verify if cRNA was at the expected the 1.2 kb average size before applying to D r a f t 8 beadchips (HumanHT-12 v.4.0, Illumina, San Diego). Beadchips were scanned with the Illumina iScan using standard conditions.
Total RNA samples were amplified and labeled with biotinylated nucleotides using a kit (Ambion, TotalPrep -96 RNA Amplification Kit). Bioanalyzer analysis was then carried out to verify if cRNA was at the expected the 1.2 kb average size before applying to D r a f t 8 beadchips (HumanHT-12 v.4.0, Illumina, San Diego). Beadchips were scanned with the Illumina iScan using standard conditions.
7
Quantitative Analysis of Piglet Intestinal Gene Expression
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Gene expression was quantified by RT-qPCR to study the expression of 56 genes in piglet jejunum samples by a customized Open Array Real-Time PCR Platform (OpenArray plate) on QuantStudio 12K Flex Real-Time PCR system (Applied Biosystems, Foster City, CA) as described by González-Solé et al. (2020) (link). For that total RNA was extracted using the Ambion RiboPure Kit (Life Technologies, Carlsbad), according to the manufacturer’s protocol. RNA was analyzed using a NanoDrop 1000A spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE) to determine if it satisfied the minimum purity and integrity standards for total RNA quality. Ten microliters of total RNA (100 ng/µL) were used for cDNA synthesis with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). The resulting cDNA was subjected to a PCR amplification followed by a real-time q-PCR reaction using the manufacturer’s TaqMan PreAmp Master Mix Kit Protocol (Life Technologies, Foster City, CA).
8
Serum RNA Extraction Protocol
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Blood samples were collected from each patient and were initially isolated into serum and cellular sediment by centrifugation at 3000 rpm for 10 minutes. The supernatant was collected and followed by further centrifugation at 12,000 rpm for 5 minutes to obtain the serum. All serum samples were stored at −80 °C before further analysis. The extraction of total RNA from 400 μL blood serum was performed using mirVana PARIS Kit (Ambion, Austin, TX) according to the manufacturer's instructions. Total RNA was eluted by 50 μL RNase-free water (Ambion, Austin, TX). The concentration and purity of the RNA solution was measured by detecting its absorbance at 260/280 and 260/230 nm with NanoDrop 1000A spectrophotometer (NanoDrop Technologies, Wilmington, DE). All the purified RNA samples were stored at −80 °C for further processing.
9
FFPE Tissue RNA Isolation Protocol
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Sixty archival FFPE cases were evaluated by two breast cancer expert pathologists. Regions of invasive carcinoma were confirmed, and different areas with more than 80% of malignant epithelial cells were selected. Four to eight μm sections were used for total RNA isolation, with MasterPure RNA Purification Kit (EPICENTRE Biotechnologies, Madison, WI, USA) according to the manufacturer's instructions with minor modifications. RNA concentrations and quality were measured using a Nanodrop 1000A spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA).
10
Sarcoma Cell Lines Characterization
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Four primary sarcoma cell lines (STS48, STS93, STS109, and STS117) derived from patients diagnosed with UPS were utilized for the in vitro experiments. Cell lines were tested and authenticated using Short Tandem Repeat (STR) profiling of the following loci for each cell line: Amelogenin, CSF1PO, D13S317, D16S539, D18S51, D19S433, D21S11, D2S1338, D3S1358, D5S818, D7S820, D8S1179, FGA, THO1, TPOX, and vWA. Cells were tested on the second passage after the initial receipt from the laboratory of RG. All in vitro experiments were performed using cells within 20 passages from the time of STR testing.
Cells were incubated at 37°C under 5% CO2 in DMEM:F12 1:1 media with 10% bovine serum. Lipofectamine-2000 (Invitrogen, Carlsbad, CA) was used to transfect cells with pre-miRs (Invitrogen), Locked-Nucleic-Acid (LNA) anti-miRs (Exiqon), or siRNAs (Qiagen). Total RNA from samples were extracted using RNeasy kits (Qiagen). Recovered RNA concentration and quality were measured using the Nanodrop 1000A spectrophotometer (Nanodrop Technologies, Wilmington, DE).
Cells were incubated at 37°C under 5% CO2 in DMEM:F12 1:1 media with 10% bovine serum. Lipofectamine-2000 (Invitrogen, Carlsbad, CA) was used to transfect cells with pre-miRs (Invitrogen), Locked-Nucleic-Acid (LNA) anti-miRs (Exiqon), or siRNAs (Qiagen). Total RNA from samples were extracted using RNeasy kits (Qiagen). Recovered RNA concentration and quality were measured using the Nanodrop 1000A spectrophotometer (Nanodrop Technologies, Wilmington, DE).
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