RNA from 1 million TNBC cells was isolated using the High Pure RNA Isolation Kit (Roche, Basel, Switzerland) and tested for quality and quantity using the NanoDropTM One/OneC (Thermo Fisher, Carlsbad, CA, USA). cDNA synthesis was performed using a FastGeneScriptase II reverse–transcription PCR kit (Nippon Genetics Europe, Dueren, Germany), and 10 ng of cDNA was used for quantitative PCR. Previously described primers for MICA (FWD: 5′-ggcgcctaaagtctgagaga-’3, REV: 5′-aaccctgactgcacagatcc-′3), MICB (FWD: 5′-ctgagaaggtggcgacgta-′3, REV: 5′-cgaagactgtggggctca-′3), ULBP1 (FWD: 5′-actgggaacaaatgctggat-′3, REV: 5′-gagaaggctccagggactg-′3), ULBP2 (FWD: 5′-ccgctaccaagatccttctg-′3, REV: 5′-gggatgacggtgatgtcatag-′3), ULBP3 (FWD: 5′-tccctggcatctgagaagag-′3, REV: 5′-cagaaaggcacagtggtgagt-′3), ULBP4 (FWD: 5′-agcacttggggagaattgac-′3, REV: 5′-cttgcagagtggaaggatcac-′3) and GAPDH (FWD: 5′-agccacatcgctcagacac-′3, REV: 5′-gcccaatacgaccaaatcc-′3) were used for quantitative PCR and were performed using Perfecta SYBR Green FastMix (Quanta Biosciences Beverly, MA, USA) measured on a LightCycler480 (Roche, Basel, Switzerland) [13 (link),21 (link)].
Nanodrop one onec
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Japan, China
The NanoDrop™ One/OneC is a spectrophotometer designed for nucleic acid and protein quantification. It provides accurate and reproducible measurements of sample concentrations using small sample volumes, typically 1-2 microliters.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (6 mentions)
Primescript rt reagent kit, by Takara Bio (4 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (4 mentions)
Applied biosystems 7900ht fast real time pcr system, by Thermo Fisher Scientific (4 mentions)
Mirneasy micro kit, by Qiagen (4 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Rneasy fibrous tissue mini kit, by Qiagen (2 mentions)
Transcriptome analysis console software, by Thermo Fisher Scientific (2 mentions)
Expression console, by Thermo Fisher Scientific (2 mentions)
Clariom s genechip, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Plant rna kit, by Aidlab (2 mentions)
Dneasy blood and tissue kit, by Qiagen (2 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Qubit rna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Lightcycler 480, by Roche (1 mentions)
High pure rna isolation kit, by Roche (1 mentions)
57 protocols using nanodrop one onec
1
Quantitative Analysis of NK Cell Ligands in TNBC
2
Profiling RNA Expression by RT-qPCR
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Total RNA was extracted from cells using Quick RNA miniprep extraction kit (Zymo Research, Irvine, CA) following the manufacturer’s instructions and quantified at A260/280 on spectrophotometer (NanoDropTM One/OneC, ThermoFisher, United Kingdom). Extracted RNA was either used for downstream analysis on the same day or stored at −80 °C for later use. cDNA was made by reverse transcription and real time PCR was performed in a single step using the Luna Universal One-Step RT-qPCR kit (New England Biolabs, Ipswich, MA, USA) following the manufacturer’s instructions with the following cycling conditions: 55 °C for 10 min; 95 °C for 1 min; 95 °C for 10 s; 60 °C for 60 s; 40 cycles. The RT-qPCR reactions were performed in triplicates per sample for two independent experiments. RNA expression of target genes (SOCS, PIAS and antiviral molecules) were expressed as fold change in relation to the expression level of the endogenous gene, β-actin using the 2−ΔΔCt relative expression formulae. All the primers that were used in this study are listed in Table 1 .
3
Intestine and Liver Gene Expression
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The total RNAs from intestine tissues (duodenum, jejunum, ileum) and liver were extracted with TRIzol reagent by following the instructions (Biomed, RA-101-02, Beijing, China). The isolated total RNAs were reversely transcribed into cDNAs using the M-MLV4 First-Strand cDNA Synthesis Kit (Takara Biotechnology, Co., Ltd., Dalian, China) after measuring the RNA concentration with a microspectrophotometer (Thermo Scientific, NanoDropTM One/OneC, Waltham, MA, USA). Real-time PCR was performed using the SYBR premix Ex Taq. II Kit (Takara Biotechnology, Co., Ltd., Dalian, China) and the results of mRNA expression were calculated via 2−ΔΔCt method and normalized to β-actin expression. The primers for superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT), nuclear factor erythroid-2-related factor 2(Nrf2), Interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), toll-like receptor4 (TLR4), zonula occludens protein 1 (ZO-1), occludin, Mucin2 (MUC2), and β-actin are listed in Table 1 .
4
Quantitative PCR Analysis of Gene Expression
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Total RNA was extracted using TRIzol reagent (Invitrogen, Waltham, MA, USA), and the concentration and quality of isolated RNA were detected by the spectrophotometer (NanoDrop™ One/OneC, Thermo Scientific). Complementary DNA (cDNA) was generated from mRNA using oligo-dT primers and PrimeScript RT reagent kit (Takara, Beijing, China). The cDNA was used as a template in RT-qPCR using TB Green Premix ExTaq (Takara) on the CFX96™ Real-Time system (Bio-Rad, CA, USA). GAPDH was used as an internal control, and the target gene expression was calculated by the 2-ΔΔCt method. The primer sequences are listed in Additional File 1 .
5
RNA Extraction from Pooled Cerebral Nuclei
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The tissue was transferred from the cryovials into beads-filled tubes (Precellys® Lysing Kit CK 14, Bertin, France). The CN of the animals of one age group were pooled in one bead tube (n = 4 animals and 8 CN per age group) without a medium. Pooling was necessary to generate enough mRNA for further analysis. Weighing of pooled CN was performed (Sartorius® Handy M160, Goettingen, Germany). The pooled CN weighed less than 20 mg, regardless of age. Therefore, following the instructions of the RNeasy Mini Kit (Qiagen®, Venlo, The Netherlands), 350 µL of RLT buffer (Qiagen®, Venlo, The Netherlands) was added per tube. These were homogenized in two homogenizer steps (Precellys 24 DUAL®, Bertin, France) at 6000 rpm for 30 s each. A total of 350 µL of ethanol 70% (Thermo Fisher Scientific®, Waltham, MA, USA) was added to the resulting emulsion. Further steps were performed according to the instructions of the RNeasy Mini Kit (Qiagen®, Venlo, The Netherlands).
Subsequently, the extracted RNA was quantified using a spectrophotometer (NanoDrop One/Onec, Thermo Fisher Scientific®, Waltham, MA, USA), and its purity (A260/A280) was determined. At postnatal day 6 (p6), animals had approximately 350 ng/mL RNA. p12 and p24 animals had about 750 ng/mL. Only RNA with an A260/A280 ratio of 2.0 ± 0.1 was used to synthesize complementary DNA (cDNA).
Subsequently, the extracted RNA was quantified using a spectrophotometer (NanoDrop One/Onec, Thermo Fisher Scientific®, Waltham, MA, USA), and its purity (A260/A280) was determined. At postnatal day 6 (p6), animals had approximately 350 ng/mL RNA. p12 and p24 animals had about 750 ng/mL. Only RNA with an A260/A280 ratio of 2.0 ± 0.1 was used to synthesize complementary DNA (cDNA).
6
Transcriptome Profiling of Cryo-Pulverized Tissues
7
RNA Extraction from Brain Regions
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Total RNA from the hippocampus, cerebellum, and cerebral cortex was extracted using Invitrogen TRIzol Reagent (Carlsbad, CA, USA), whereas RNA from the liver and cell culture was extracted using NucleoSpin RNA Plus (Macherey-Nagel), following the manufacturer’s protocols. RNA quality was determined using NanoDrop™ One/OneC (Thermo Fisher Scientific), where a 260/280 ratio of >2.0 and 260/230 ratio between 2.0–2.2 were considered as pure RNA.
8
Transcriptome Profiling of Cryo-Pulverized Tissue
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Total RNA was isolated from cryo-pulverized tissue from the I zone using a Qiagen RNeasy Fibrous Tissue Mini Kit according to the manufacturer’s protocol. Extracted RNA was quantified spectrophotometrically with NanoDrop One/Onec (Thermo Fisher Scientific), and RNA quality was assessed using an Agilent 2100 Bioanalyzer. Transcriptome profiling was performed using the Affymetrix Clariom S GeneChip according to the manufacturer’s instructions. Raw data were analyzed using Affymetrix Expression Console and Transcriptome Analysis Console software prior to downstream analysis. Statistically significant differentially expressed genes (DEGs) were identified using an expression fold change (FC) > 1.7 or < − 1.7 and a false discovery rate of p < 0.08. Semi-quantitative real-time polymerase chain reaction (PCR) of select DEGs was performed to confirm the expression fold changes measured using the Affymetrix Clariom S GeneChip (Figs. S2 and S3).
9
RNA Quality and Quantity Assessment
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The RNA quality and quantity were assessed using the NanoDrop spectrophotometer (NanoDrop One/Onec, Thermo Scientific) after extraction of total RNA. Total RNA was converted to complementary DNA (cDNA) using total isolated RNA, random hexamer primer and reverse transcriptase according to manufacturer’s protocol (Thermo Scientific RevertAid First Strand cDNA Synthesis Kit, USA).
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The RNA quality and quantity were assessed using the NanoDrop spectrophotometer (NanoDrop One/Onec, Thermo Scientific) after extraction of total RNA. Total RNA was converted to complementary DNA (cDNA) using total isolated RNA, random hexamer primer and reverse transcriptase according to manufacturer’s protocol (Thermo Scientific RevertAid First Strand cDNA Synthesis Kit, USA).
10
Microplate-based MDA Quantification
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The expression of MDA was detected with a micro MDA assay kit (Solarbio). Briefly, cells were incubated with thiobarbituric acid (TBA, 2.8% w/v) at 95°C for 60 min and then centrifuged at 4°C, 2,500 × g for 10 min. The reactive complexes were then detected using a NanoDrop One/Onec spectrophotometer at 532 nm (Thermo Fisher Scientific).
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