Total RNA was extracted from each sample using the TRIzol reagent (Invitrogen, Carlsbad, USA) and DNase I was then used to remove DNA (Takara, Dalian, China). The quality, quantity, and integrity of the total RNA were evaluated using Nanodrop (IMPLEN, CA, USA), Qubit 2.0 (Life Technologies, CA, USA), and Aglient 2100 (Agilent Technologies, CA, USA). Briefly, the first cDNA chain was synthesized using random hexamers as a six-base random primer, and the second cDNA chain was synthesized by adding a buffer solution, dNTPs, RNase H, and DNA polymerase I. The purified double-stranded cDNAs were terminal repaired, tailed, and sequenced. Then, the fragment size was selected by AMPure XP (Beckman Coulter, Brea, CA, USA) beads and the cDNA library was obtained by PCR enrichment. Finally, libraries were loaded on Illumina/Solexa HiSeq2000 platform with a sequenced read length set to PE100.
Nanodrop
Manufactured by Implen
Sourced in Germany, United States
The Nanodrop is a spectrophotometer designed for the measurement of small sample volumes. It allows for the rapid and accurate quantification of nucleic acids, proteins, and other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (11 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (6 mentions)
Qubit 2, by Thermo Fisher Scientific (3 mentions)
Agilent 2100, by Agilent Technologies (3 mentions)
Lightcycler 480, by Roche (3 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (2 mentions)
Revertra ace qpcr rt kit, by Toyobo (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Total rna extraction kit, by Tiangen Biotech (2 mentions)
Peqgold trifast, by Avantor (2 mentions)
Trizol, by Merck Group (2 mentions)
Advantage rt for pcr kit, by Takara Bio (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Dnase 1, by Takara Bio (1 mentions)
Ampure xp, by Beckman Coulter (1 mentions)
Aglient 2100, by Agilent Technologies (1 mentions)
Rq1 rnase free dnase 1, by Promega (1 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
Revertaid first stand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
52 protocols using nanodrop
1
RNA Extraction and RNA-Seq Library Preparation
2
Comprehensive RNA Extraction and Analysis
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Total RNA was extracted using TRIzol reagent according to the protocol of the provider company (Invitrogen, USA).
The concertation of isolated RNA and its integrity were analyzed via NanoDrop (IMPLEN, Germany) and agarose gel, respectively.
RNA samples were incubated with RQ1 RNase-free DNase I (Promega) to remove possible DNA contaminations.
To confirm the removal of DNA contamination, the extracted RNA samples were used as the template in PCR reaction with genomic DNA as control.
the absence of PCR products in DNase-treated RNA samples indicated the absence of genomic DNA. cDNA was generated using a cDNA Reverse Transcription kit (Viva 2-step RT-PCR kit).
Specific primers for (-)-limonene synthase (LS), (-)-menthone reductase (MR) and actin (internal control) were
designed by Primer Quest software (Supplementary Table 1 ).
The quantitative real-time PCR was carried out using Step One PlusTM (ABI 7500 Applied Biosystems).
The RT-PCR program included an initial denaturation (3 min) at 95 °C, followed by 40 cycles of denaturation (95 °C, 30 sec), annealing (60 °C, 30 sec),
and elongation (72 °C, 30 sec), and a final elongation step (72 °C, 5 min). After the determination of the amplification of efficiency (E value),
the relative expression of samples was analyzed by the Livak method using the REST 2009 Software ( 31 (link)
).
The concertation of isolated RNA and its integrity were analyzed via NanoDrop (IMPLEN, Germany) and agarose gel, respectively.
RNA samples were incubated with RQ1 RNase-free DNase I (Promega) to remove possible DNA contaminations.
To confirm the removal of DNA contamination, the extracted RNA samples were used as the template in PCR reaction with genomic DNA as control.
the absence of PCR products in DNase-treated RNA samples indicated the absence of genomic DNA. cDNA was generated using a cDNA Reverse Transcription kit (Viva 2-step RT-PCR kit).
Specific primers for (-)-limonene synthase (LS), (-)-menthone reductase (MR) and actin (internal control) were
designed by Primer Quest software (
The quantitative real-time PCR was carried out using Step One PlusTM (ABI 7500 Applied Biosystems).
The RT-PCR program included an initial denaturation (3 min) at 95 °C, followed by 40 cycles of denaturation (95 °C, 30 sec), annealing (60 °C, 30 sec),
and elongation (72 °C, 30 sec), and a final elongation step (72 °C, 5 min). After the determination of the amplification of efficiency (E value),
the relative expression of samples was analyzed by the Livak method using the REST 2009 Software ( 31 (link)
).
3
Characterizing Inflammatory Gene Expression in HepG2 Cells
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HepG2 cells (106) were treated with 0.3 mM FFAs for 2, 4, and 8 h and subsequently harvested for total RNA isolation. The total RNA was extracted using TRIzol reagent (Life Technologies, Carlsbad, CA, USA), and quantified using NanoDrop (Implen, Munich, Germany). One microgram of RNA was reversely transcribed to cDNA using RevertAid First Stand cDNA Synthesis Kit (K1622, Fermentas, Waltham, MA, USA).The quantitative, real-time RT-PCR (qRT-PCR) was performed with SsoFast EvaGreen Supermix reagent (Bio-Rad, Hercules, CA, USA) using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). The thermal cycle program was set as follows: 95 °C for 3 min, 39 cycles for amplification at 98 °C for 5 s and 60 °C for 5 s. The following primer pairs were used for qRT-PCR analysis: human interleukin-8 (IL-8; forward: 5′-CTTTCAGAGACAGCAGAG-3′ and reverse: 5′-CTAAGTTCTTTAGCACTCC-3′), human tumor necrosis factor-alpha (TNF-α forward: 5′-CCTGTGAGGAGGACGAAC-3′ and reverse: 5′-CGAAGTGGTGGTCTTGTTG-3′), and the housekeeping Actin gene (forward primer: 5′-GAGATGCGTTGTTACAGGAA-3′ and reverse: 5′-GCATTACATAATTTACACGAAAGC-3′). The relative mRNA expression was normalized to the control actin gene.
4
Quantitative RNA Expression Analysis
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Total RNA extraction was performed using RNeasy plus mini kits (Qiagen, Germany) following the manufacturer’s instructions. Subsequently, the quality of the extracted RNA was determined using the NanoDrop instrument (Implen, Germany). Then, complementary DNA was synthesized using the ReverTra Ace qPCR RT Kit (Toyobo, Japan). Finally, the qRT-PCR was performed on the QuantStudio 6 Flex System (Life Technologies). The specific primer sequences are shown in Supplementary Table S1 . GAPDH was used as the internal control, and the expression of the targeted genes was normalized to GAPDH.
5
Isolating RNA from Frozen Plant Roots
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To isolate the RNA from the frozen roots, the tissues were ground with mortar and pestle using liquid nitrogen. Extraction of RNA from the roots was done using the PuroZOL RNA isolation reagent (BioRad Laboratories, California, USA). DNA was eliminated using DNAse digestion (Thermo Fisher Scientific, USA). RNA quality and quantity were determined by Nanodrop (Implen GmbH, Munich, Germany).
6
Hamstring Tendon Stromal Cell RNA Extraction
7
Purification of ABCB6 Membrane Protein
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For the protein purification of the ABCB6, two liters of transfected HEK293F cells were harvested by centrifugation at 3000× g. Cell pellets were resuspended in lysis buffer containing 20 mM Hepes, pH 7.4, and 150 mM NaCl, a protease inhibitor cocktail tablet (Roche, 1:1000), and lysed by sonication for 5 min. The cell membrane was pelleted after a 100,000× g ultracentrifugation for 1 h. The membrane was resuspended in a buffer containing 20 mM Hepes, pH 7.4, 150 mM NaCl, 2 mM DTT, and 1% (w/v) C12E9 for 1.5 h with gentle rotation at 4 °C. After ultracentrifugation at 100,000× g for 15 min, the supernatant was incubated with Strep-Tactin Sepharose (IBA) for 0.5 h with gentle rotation at 4 °C. The resin was washed extensively with wash buffer containing 20 mM Hepes, pH 7.4, 150 mM NaCl, and 0.01% (w/v) C12E9. The target protein was eluted with wash buffer plus 5 mM d -desthiobiotin (IBA) and concentrated to a final volume of approximately 100 μL. The final protein was applied to size-exclusion chromatography (Superpose-6 10/300 GL, GE Healthcare) in a buffer containing 20 mM Hepes, pH 7.4, 150 mM NaCl, and 0.01% C12E9. The peak corresponding to the ABCB6 or ABCB6 mutant was collected and protein concentrations in detergent micelles were measured by absorbance at 280 nm using a NanoDrop (Implen).
8
RNA Extraction from Chest Muscles
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We used the method for RNA extraction referred to in Xue et al. [20 (link)] where total RNA from the chest muscles was isolated using the TRIzol total RNA extraction kit (Invitrogen, Carlsbad, CA, USA), in accordance with the manufacturer’s instructions. RNA quality was checked with a 1% agarose gel. RNA concentration and purity were determined using NanoDrop (IMPLEN, Los Angeles, CA, USA). Sample integrity was measured using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA).
9
Fungal/Bacterial DNA Extraction Protocol
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For DNA extraction, the grains were isolated from the infected tissue using a sterile surgical blade and transferred to a sterile Eppendorf tube (1.5 mL), containing 10 metal beads and 700 μL Bashingbead buffer (Zymo DNAfungal/bacteria extraction kits, Irvine, CA, USA). The mixture was then lysed using a Tissuelyser II (Qiagen, Germany) for 3 min at 30 hertz. The supernatant was removed and added onto a Zymo‐Spin III‐F filter column and spun down for 1 min at 8000 g. From then onwards, DNA was isolated via the protocol of the manufacturer. The concentration and purity of the extracted DNA were measured using a NanoDrop spectrophotometer (Implen, Germany). The obtained DNA was stored at −20°C until further use.
10
Transcriptome analysis of male and female antennae
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Total RNA was extracted from male and female antennae with TRIZOL reagent using the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). A nanodrop (IMPLEN, CA, USA), Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA) and Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA) were used to detect the purity, concentration and integrity of RNA samples, and RNA degradation and contamination were monitored on 1% agarose gels to ensure the quality of the samples used for transcriptome sequencing.
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