Muscle tissue of a 5-month-old female P. monodon was collected from the Shrimp Genetic Improvement Center (SGIC, Surat Thani, Thailand), immediately frozen in liquid nitrogen, and stored at −80 °C until use. Frozen muscle tissue was pulverized in liquid nitrogen and genomic DNA was extracted using a Genomic Tip 100/G kit (Qiagen, USA) as previously described (Angthong et al., 2020 (link)). DNA quantity was measured using a NanoDrop ND-8000 spectrophotometer and a Qubit dsDNA BR Assay kit (Invitrogen, USA) using Qubit fluorometer. The DNA quality and integrity were visualized under pulsed-field gel electrophoresis at 80 volts for 9 h in 0.5x KBB buffer (51 mM Tris, 28 mM TASP, 0.08 mM EDTA, pH 8.7) (Sage Science, USA) containing SYBR Safe DNA gel staining (Invitrogen, USA).
Nanodrop nd 8000 spectrophotometer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The NanoDrop ND-8000 spectrophotometer is a laboratory instrument used to measure the concentration and purity of nucleic acid and protein samples. It utilizes a patented sample retention system that requires only 1-2 microliters of sample to perform accurate measurements.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (13 mentions)
Rneasy mini kit, by Qiagen (8 mentions)
Trisure, by Meridian Bioscience (5 mentions)
Paxgene blood mirna kit, by Qiagen (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Paxgene blood rna tube, by Preanalytix (4 mentions)
Fastdna spin kit, by MP Biomedicals (3 mentions)
Quantitect reverse transcription kit, by Qiagen (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Mirneasy kit, by Qiagen (3 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Sybr safe dna gel staining, by Thermo Fisher Scientific (2 mentions)
Genomic tip 100 g kit, by Qiagen (2 mentions)
Cls tc, by MP Biomedicals (2 mentions)
Rnalater, by Thermo Fisher Scientific (2 mentions)
Isolate 2 rna mini kit, by Meridian Bioscience (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Trizol reagent, by Takara Bio (2 mentions)
Mirneasy mini kit, by Qiagen (2 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (2 mentions)
67 protocols using nanodrop nd 8000 spectrophotometer
1
Genomic DNA Extraction from Shrimp Muscle
2
Genomic DNA Extraction from Shrimp Muscle
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The muscle of a 5-month-old female P. monodon was collected from the Shrimp Genetic Improvement Center (SGIC, Surat Thani, Thailand), immediately frozen in liquid nitrogen and stored at -80 °C until use. Frozen muscle was pulverized in liquid nitrogen and genomic DNA was extracted using a Genomic Tip 100/G kit (Qiagen, USA). DNA quantity was measured using a NanoDrop ND-8000 spectrophotometer and a Qubit dsDNA BR Assay kit (Invitrogen, USA) using Qubit fluorometer. The DNA quality and integrity were visualized under pulsed-field gel electrophoresis at 80 volts for 9 h in 0.5x KBB buffer (51 mM Tris, 28 mM TASP, 0.08 mM EDTA, pH 8.7) (Sage Science, USA) containing SYBR Safe DNA gel staining (Invitrogen, USA).
3
DNA Extraction from Apple Wash Solution
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The DNA was extracted from the pellet obtained from 50 mL of the apple washing solution (see Section 3.2 ) and the extraction was performed using the FastDNA™ SPIN Kit (MP Bio-medicals, IIIkirch, France). The DNA extraction was conducted following the method illustrated by Al Riachy et al. [58 (link)]. The first step consisted of the resuspension of the pellet in 1 mL of the lysing solution CLS-TC (MP Biomedicals, Germany), included in the kit, and then its transfer to the lysing matrix A tube. The Garnet matrix and a 1/4″ ceramic sphere were incorporated into the latter to aid in cell lysis. Subsequently, the samples underwent grinding using the FastPrep® Instrument for a duration of 50 s at a speed of 6 m/s. The remaining steps of the extraction followed the manufacturer’s specifications (MP Bio-medicals, IIIkirch, France). Finally, the DNA was resuspended in 50 µL of DNase-free water (DES), and its purity ratio and concentration were assessed using a Nanodrop ND 8000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
4
FFPE RNA Extraction and Quality Control
5
Extraction and Purification of Rice Genomic DNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GM rice G281 event and its recipient line were grown in a greenhouse in Shanghai Jiao Tong University, China, and fresh leaves were sampled and ground into powder in liquid nitrogen for DNA extraction. Rice genomic DNA was extracted and purified using a Plant Genomic DNA Kit (Cat. no. DP305-3; TIANGEN Company, China). The quality and quantity of extracted DNA was evaluated with a Nanodrop ND-8000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and by 1% agarose gel electrophoresis, respectively. Purified DNA samples were stored and used for PCR, droplet digital PCR (ddPCR), and PE library construction.
6
Detailed Jejunal RNA Extraction Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For RNA extraction, the jejunal tissues were carefully flushed with phosphate-buffered saline and immediately placed in RNAlater (Invitrogen, Carlsbad, CA) and stored at −20°C until further use. Approximately 80 mg of the sample tissue was immersed in 1 mL TRIsure, (Bioline, Sydney, Australia) homogenized with an IKA ultra-turrax homogenizer (IKA-Work, Staufen, Germany). Total RNA was extracted as per the manufacturer's instructions. Total RNA was purified using ISOLATE II RNA Mini Kit (Bioline, Sydney, Australia) and a DNase I digestion step was included to remove any remaining genomic DNA as per the manufacturer's instructions. A NanoDrop ND-8000 spectrophotometer (Thermo Fisher Scientific, Waltham) and the Agilent 2,100 Bioanalyzer (Agilent Technologies, Inc., Waldbronn, Germany) were used to determine the concentration and RNA integrity (RNA Integrity Number, or RIN), respectively. Purified total RNA was stored at −80°C until further use.
7
Transcriptome Profiling of Apple Scald
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
From the fruit of each condition/stage, the total RNA was extracted with the Spectrum Plant total RNA kit (Sigma-Aldrich, St. Louis, MO, USA). RNA was quantified and controlled using a NanoDrop ND8000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA), respectively. To capture the effect of each treatment in modulating the development of scald, the control sample together with 1-MCP and lovastatin samples, collected after 4 months of cold storage + 5 days of shelf life at 20 °C, were employed to perform a genome-wide RNA-sequencing assay. For each replicate/sample, 3 μg of RNA (isolated from three replicates per sample) were used for the preparation of RNA-Seq library using the NEB Next Ultra II kit (BioLabs Inc., New England, following the manufacturer’s instruction). The libraries obtained here were quantified using qPCR and controlled with the DNA 1000 series II chip bioanalyzer (Agilent, Santa Clara, CA, USA). The libraries were then sequenced using an Illumina NextSeq 500 (read length of 75 bp) at the Functional Genomic Lab at the University of Verona (Italy). RNA-Seq raw data are freely available at the Gene Expression Omnibus (GEO) database with the following accession number GSE136374 .
8
Quantification of SHP gene expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from 10-µm slices of FFPE samples from potentially eligible patients (n=12) using a Qiagen RNeasy® FFPE kit (Qiagen, Inc.) following the manufacturer's protocol. RNA quantitation and purity were measured using a NanoDrop™ ND-8000 spectrophotometer (Thermo Fisher Scientific, Inc.).
cDNA was synthesized by RT using SuperScript™ II Reverse Transcriptase (cat. no. 18064; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCRs were carried out using Rotor-Gene SYBR-Green PCR kits (cat. no. 204074; Qiagen, Inc.) in a Rotor-Gene Q 2plex system (cat. no. 9001620; Qiagen, Inc.). The samples were amplified for 40 cycles as follows: 95°C for 10 sec and 60°C for 30 sec. To analyze the qPCR data, relative quantification was performed using the 2−ΔΔCq method with human GAPDH as the internal control gene; data are expressed as relative fold-changes (32 (link)). The sequences of human SHP and GAPDH primers were as follows: SHP forward, 5′-TCCTCTTCAACCCCGATGTG-3′ and reverse, 5′-CAGGGTTCCAGGACTTCACAC-3′: GAPDH forward, 5′-TCGGAGTCAACGGATTTGGT-3′ and reverse, 5′-TTCCCGTTCTCAGCCTTGAC-3′.
cDNA was synthesized by RT using SuperScript™ II Reverse Transcriptase (cat. no. 18064; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCRs were carried out using Rotor-Gene SYBR-Green PCR kits (cat. no. 204074; Qiagen, Inc.) in a Rotor-Gene Q 2plex system (cat. no. 9001620; Qiagen, Inc.). The samples were amplified for 40 cycles as follows: 95°C for 10 sec and 60°C for 30 sec. To analyze the qPCR data, relative quantification was performed using the 2−ΔΔCq method with human GAPDH as the internal control gene; data are expressed as relative fold-changes (32 (link)). The sequences of human SHP and GAPDH primers were as follows: SHP forward, 5′-TCCTCTTCAACCCCGATGTG-3′ and reverse, 5′-CAGGGTTCCAGGACTTCACAC-3′: GAPDH forward, 5′-TCGGAGTCAACGGATTTGGT-3′ and reverse, 5′-TTCCCGTTCTCAGCCTTGAC-3′.
9
Extraction and Analysis of Jejunal mRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total mRNA from jejunum samples was extracted by Trizol reagent (TaKaRa Biotechnology Co. Ltd., Dalian, Liaoning, China). The extracted RNA was treated with RNeasy Mini Kit (Qiagen, Cat. No. 74104) corresponding to the guidelines of the manufacturer. The amount and pureness of the total RNA were detected by NanoDrop ND-8000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Corresponding DNA (cDNA) gained by reversal-transcription of the isolated RNA samples utilizing RevertAidTM H Minus kits (Fermentas Life Science, Pittsburgh, PA, USA). One μL of this cDNA was mixed with 2x maxima SYBR Green PCR mix (12.5 μL) and RNase free water (10.5 μL); later 0.5 μL of each forward and reverse primer for the selected genes were added. The sequences of the primers of genes encoding amino acids and peptides transporters are depicted in Table 4 . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as control gene.
10
Jejunal RNA Extraction and cDNA Synthesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For RNA extraction, the jejunal tissues were rinsed with chilled phosphate-buffered saline and immediately placed in RNAlater (Invitrogen, Carlsbad, CA), stored at 4 °C for 3 to 5 h and then stored at −20 °C until further use. Approximately 80 mg of the sample tissue was homogenized in 1-mL TRIsure (Bioline, Sydney, Australia) using an IKA T10 basic Homogenizer (Wilmington, NC, USA) and the total RNA was extracted as per the manufacturer's instructions. Total RNA was then purified using a ISOLATE II RNA Mini kit (Bioline, Sydney, Australia) as per the manufacturer's instructions and a DNA digestion step using DNase I was included to eliminate genomic DNA. A NanoDrop ND-8000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Waldbronn, Germany) were used to determine the concentration and RNA integrity (RNA integrity number, RIN) respectively. Purified RNA from each sample was reverse-transcribed to cDNA with the SensiFAST cDNA synthesis kit (Bioline, Sydney, Australia) as per the manufacturer's instructions. A Rotorgene 6000 real-time PCR instrument (Corbett, Sydney, Australia) was used to convert the RNA into cDNA. Synthesized cDNA samples were diluted 1:10 with nuclease-free water and stored at −20 °C until used.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!