Cells were resuspended in nuclear extraction buffer (Tris 10 mM pH 7.5; 10 mM NaCl; 5 mM MgCl2; 0.5% NP-40), incubated at 4 °C for 5 min and spin down 5 min at 500g. The cytosolic fraction was discarded. The pellet, corresponding to the nuclear fraction, was washed twice in the nuclear extraction buffer, resuspended in Trizol and passed through a 20-G needle five times. RNA extraction was done with mi-RNeasy kit (QIAGEN #217004) and RNA concentrations were determined with a NanoDrop apparatus (NanoDrop Technologies, Inc.). Before cDNA synthesis, nuclear RNA were treated with DNaseI, RNase free (Thermo Scientific #EN0525) in reaction buffer with MgCl2 according to the manufacturer's protocol.
Nanodrop apparatus
Manufactured by Thermo Fisher Scientific
Sourced in United States, France
The NanoDrop apparatus is a spectrophotometric device used for the quantification and analysis of small volume samples. The core function of the NanoDrop is to measure the absorbance of ultraviolet and visible light by molecular samples, enabling the determination of concentration and purity of biomolecules such as nucleic acids and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (7 mentions)
Iscript cdna synthesis kit, by Bio-Rad (4 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Sybr green supermix, by Bio-Rad (3 mentions)
Te buffer, by Merck Group (3 mentions)
Proteinase k, by Roche (3 mentions)
Rnase a, by Roche (3 mentions)
Mirneasy kit, by Qiagen (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Cfx manager 2, by Bio-Rad (2 mentions)
Lysozyme, by Merck Group (2 mentions)
Ribavirin, by Merck Group (2 mentions)
Glutathione sepharose matrix, by GE Healthcare (2 mentions)
Poly da dt po833, by Merck Group (2 mentions)
Cyclic diguanosine monophosphate cyclic di gmp, by Biolog (2 mentions)
Recombinant ifn a d, by PBL Assay Science (2 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (2 mentions)
Qiaamp dna stool mini kit, by Qiagen (2 mentions)
Lightcycler 96 apparatus, by Roche (2 mentions)
52 protocols using nanodrop apparatus
1
Nuclear RNA Extraction Protocol
2
RNA Isolation and Purification Procedure
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Total RNA isolation was performed using miRNEasy Kit from (Qiagen, #217004) according to the manufacturer's instructions. RNA concentrations were determined with a NanoDrop apparatus (NanoDrop Technologies, Inc.), and RNA integrity was verified using Agilent RNA 6000 Nano Kit (Agilent, #5067–1511) and apparatus. Before complementary DNA synthesis, RNA were treated with DNaseI, RNase free (Thermo Scientific, #EN0525) in reaction buffer with MgCl2 according to the manufacturer's protocol.
3
Polygyne S. invicta Colony Sampling
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Three polygyne S. invicta colonies were collected from South China Agricultural University (SCAU, Guangzhou, China) campus in May 2014. Colonies were reared inside plastic boxes (50 cm × 40 cm × 15 cm) under controlled laboratory conditions of 25 ± 2°C, 75 ± 5 relative humidity and 12:12 (L:D) h light. Colonies were fed with Tenebrio molitor larvae and 25% sucrose water every other day. Two groups of workers were sampled: foragers from outside the nest (foraging in the food area) and nurses from inside the nest (caring for the brood). We collected 10 foragers and nurses from each of the three colonies at around 10:00 am for most of the foragers re observed outside the nest around this time. Workers were directly frozen in liquid nitrogen. The total of thirty (foragers or nurses) were pooled and were kept at −80°C until further processing. RNA of whole ant bodies was extracted using RNAiso Plus (TaKaRa, Code number 9108). DNase I (RNase-free, TaKaRa) was used to digest genomic DNA from the RNA solution. NanoDrop apparatus (NanoDrop Technologies, Inc.) and Agilent 2100 bioanalyzer (Agilent Technologies) were used to quantify and check for integrity of extracted RNA.
4
Comparative RT-PCR Analysis of Gene Expression
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Two different RT-PCRs were used in this study, real-time PCR and semi-quantitative PCR. Total RNA from tissues and cells was isolated by TRIzol reagent, and RNA concentrations were determined with a NanoDrop apparatus (NanoDrop Technologies, Wilmington, DE, USA). Two milligrams of total RNA was reverse transcribed using a First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Real-time PCR was performed using SYBR Green master mix (Applied Biosystems, Foster City, CA, USA) and a 7500 Fast Real Time PCR system (Applied Biosystems, Foster City, CA, USA). Expression levels were normalized to those of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primers used for real-time PCR are shown in Table 1 . The relative gene expression was calculated using the comparative cycle threshold (2−DDCt) method. The parameter for semi-quantitative PCR was 4 min at 94 °C followed by 26 cycles of 45 s at 94 °C, 30 s at 60 °C, 45 s at 72 °C and a final extension of 5 min at 72 °C. PCR products (10 µL) were used to detect the expression.
5
Total RNA Extraction and Evaluation
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Total RNA was extracted and isolated using TRIzol (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. RNA concentrations were determined with a NanoDrop apparatus (NanoDrop Technologies, Inc., and 1 ng per sample was used for the assays. RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The RNA integrity number (RIN) was determined using the RIN algorithm of the Agilent 2100 analyzer (Agilent Technologies, Palo Alto, CA, USA). RNAs with a RIN ≥ 7.0 and 28S/18S > 0.7 were accepted for qPCR analysis.
6
Phage DNA Extraction and Restriction Analysis
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Extraction of the phage genomic DNA was performed using the Wizard® DNA Clean-Up System (Promega, Madison, WI, USA), following manufacturer recommendations. The exact determination of phage genomic DNA concentrations was carried out using a NanoDrop apparatus (Nanodrop Technologies Inc., Wilmington, USA), following the instructions provided by the manufacturer. Phage DNA preparations were digested with HindIII and BamHI restriction endonucleases (New-England Biolabs, MA, USA) for 8–10 hours. Restriction fragments were separated by electrophoresis in 0.75% agarose gel, stained with ethidium bromide and visualized under ultra-violet light to roughly estimate the genome size.
7
Lipid Nanoparticle Transfection of PD-L1 siRNA
8
Microglial Cell RNA Extraction and qPCR Analysis
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Total RNA from microglial cell cultures was extracted with the RNeasy mini kit according to the manufacturer's instructions (Qiagen, Courtaboeuf, France). RNA quality and concentration were assessed by spectrophotometry with the NanoDrop™ apparatus (Thermo Scientific, Wilmington, Del., USA). Total RNA (1-2 µg) was subjected to reverse transcription using the iScript™ cDNA synthesis kit (Bio-Rad, Marnes-la-Coquette, France). qPCR was performed in duplicate for each sample using SYBR Green Supermix (Bio-Rad) for 40 cycles with a 2-step program (5 s of denaturation at 96°C and 10 s of annealing at 60°C). Amplification specificity was assessed with a melting curve analysis. Primers were designed using Primer3 software. Sequences and their NCBI references are given in online supplementary table 1 (for all online suppl. material, see www.karger.com/doi/10.1159/000370031 ). The relative expression of genes of interest was determined relative to the expression of the reference gene, GAPDH (glyceraldehyde 3-phosphate dehydrogenase). Analyses were performed with the Bio-Rad CFX Manager 2.1 software.
9
Optimized DNA Isolation from Stool Samples
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A detailed DNA isolation protocol was presented in our team’s previous studies16 (link). The frozen samples were thawed, precisely weighed (about 0.1 g of stool sample was used) and homogenized in 0.1 ml of saline. After lysis of bacterial and fungal cells with lysozyme (Sigma-Aldrich, Poznań, Poland) (1 mg/ml) and lysostaphin (Sigma-Aldrich, Poznań, Poland) (0.1 mg/ml), samples were incubated at 37 °C for 20 min. Next, 200 μl 75 mM NaOH (Avantor Performance Materials, Gliwice, Poland) was added and samples were incubated at 95 °C for 10 min. After incubation, probes were microcentrifuged (12 000 rpm, 10 min), supernatants were removed, and pellets were resuspended in 500 μl buffer supplemented with β-mercaptoethanol (Sigma-Aldrich, Poznań, Poland). For each sample, lyticase (Sigma-Aldrich, Poznań, Poland) was added (0.1 mg/ml). Probes were incubated at 37 °C for at least 30 min and microcentrifuged (12 000 rpm, 10 min). The next steps of DNA extraction were carried out according to Genomic Mini AX Stool Spin Kit (A&A Biotechnology, Gdańsk, Poland) procedure.
DNA concentration and purity was controlled spectrophotometrically using a NanoDrop apparatus (Thermo Fisher Scientific).
DNA concentration and purity was controlled spectrophotometrically using a NanoDrop apparatus (Thermo Fisher Scientific).
10
RNA Extraction and Purification Protocol
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RNA was extracted with TRIzol reagent (Ambion) and purified with the Direct-zol RNA MiniPrep kit (Zymo Research). DNase I treatment was performed on the Direct-zol column. RNA concentration was determined with a Nanodrop apparatus (Thermo Fisher Scientific) and its integrity (size distribution) with a Bioanalyzer 2100 (Agilent).
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