Total RNA was isolated using a previously described protocol (7 (link)) for the PureLink FFPE total RNA isolation kit (Invitrogen). Briefly, cells were sorted into the melting buffer containing 1600 U/mL RNase inhibitor (Bioline) and 10 mM DTT (Sigma-Aldrich Ltd.) and stored at −80°C before proceeding to the proteinase K treatment for 15 min at 60°C. Subsequently the manufacturers instructions were followed, including the optional DNase digestion. The RNA was further cleaned using the RNeasy Mini Kit RNA Cleanup protocol (Qiagen). RNA concentrations were measured using the NanoDrop 2000 (Thermo Scientific) and RNA integrity assessed using the 2100 Bioanalyser instrument (Agilent Technologies, Inc.).
2100 bioanalyser instrument
Manufactured by Agilent Technologies
Sourced in United States, United Kingdom
The 2100 Bioanalyzer is an automated electrophoresis system that analyzes and quantifies DNA, RNA, and protein samples. It provides rapid, sensitive, and reproducible results for a variety of applications, including gene expression analysis, genomic DNA QC, and microRNA profiling.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Purelink ffpe total rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Rnase inhibitor, by Meridian Bioscience (1 mentions)
Rneasy mini kit rna cleanup protocol, by Qiagen (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Bioanalyzer high sensitivity rna analysis kit, by Agilent Technologies (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Ssoadvanced preamp supermix, by Bio-Rad (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Facsaria 3, by BD (1 mentions)
Cfx maestro software, by Bio-Rad (1 mentions)
Cfx384 touch real time pcr detection system, by Bio-Rad (1 mentions)
Ovation ultralow library system v2 kit, by Tecan (1 mentions)
Rnaeasy plus mini kit, by Qiagen (1 mentions)
Kapa library quantification kit, by Agilent Technologies (1 mentions)
Kapa hyper prep kit, by Illumina (1 mentions)
Nextseq, by Illumina (1 mentions)
Dna 7500 chip, by Agilent Technologies (1 mentions)
6 protocols using 2100 bioanalyser instrument
1
FFPE Total RNA Isolation Protocol
2
Transcriptomic Analysis of Zebrafish Cell Populations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Embryos of Tg(flt1:YFP) and Tg(abcc9:Gal; UAS:GFP) were collected at 3 dpf and screened as described in Figure 3D , dissociation was performed as previously described (Kartopawiro et al., 2014 (link)). The dissociated cells were sorted using a fluorescence activated cell sorting (FACS) Aria III (BD Biosciences) into 300 μl TRIzol LS Reagent (Thermo Fisher). Total RNA was extracted using the Quick-RNA Microprep kit (Cambridge Bioscience) following the manufacturer’s instructions. RNA quality and concentration were determined using 2100 Bioanalyser Instrument (Agilent) together with Bioanalyzer High Sensitivity RNA Analysis Kit (Agilent). One ng of RNA template was subjected to cDNA synthesis using SuperScript VILO cDNA Synthesis Kit (Thermo Fisher). The synthesized cDNAs were amplified in parallel using SsoAdvanced PreAmp Supermix (Biorad), and both of the amplified samples were included for further analysis. The qPCR analysis was performed using the primers in Table S1 on CFX384 Touch Real-Time PCR Detection System (BioRad). Data were analysed using the CFX Maestro Software (BioRad). The geometric average of rpl13 and β-actin or kdrl and β-actin expression was used as a reference to calculate relative gene expression of target genes with the ddCT method and the values were presented as log or normalized log fold. Primer sequences listed in the below table.
3
Ancient DNA Extraction from Petrous Bone
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The petrous bone was treated with ultraviolet light for 20 min before the DNA extraction process. A dental drill was used for extremely careful sampling of bone powder from the petrous bone in the ancient DNA facilities of the National Research Center Kurchatov Institute (Moscow, Russia). After collection, the extracted sample was immediately used for historical DNA extraction in the same facility. Historical DNA from the H. gigas museum specimen was extracted from the bone powder following the silica beads methodology. This method is based on several sequential steps:
Historical DNA isolation in buffer containing ethylenediaminetetraacetic acid (EDTA) and proteinase K;
DNA enrichment on silica beads in the binding buffer; and
DNA washing in ethanol and DNA elution.
4
Paraquat-Induced Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We used the same three isofemale lines (S29, W120, and MT47) for our analysis of gene expression. For each of two biological replicates, fifteen 4- to 7-day-old-females were exposed for 24 h to medium supplemented with paraquat (20 mM) or without paraquat (i.e., a total of 12 samples). Flies were dissected on ice in a phosphate buffer saline solution to remove gonads, and the remaining somatic tissue was frozen in liquid nitrogen and stored at −80 °C.
We used the RNAeasy Plus Mini Kit (Qiagen) to extract total RNA from the somatic tissues, following the protocol provided by manufacturer. Samples were treated with DNAse (ref. AM2224, AMbion) according to the manufacturer’s instructions and stored at −80 °C. RNA amount and quality was estimated using Qubit (Thermo Fisher Scientific) and the 2100 Bioanalyser instrument (Agilent). RNA libraries and sequencing were performed on the GenomEast platform, a member of the ‘France Génomique’ consortium (ANR-10-INBS-0009). Libraries were constructed using the TruSeq Stranded mRNA Library Prep Kit following the manufacturer’s recommendations. The libraries were sequenced on Illumina High HiSeq 4000 with paired-end 100 base pair long reads.
We used the RNAeasy Plus Mini Kit (Qiagen) to extract total RNA from the somatic tissues, following the protocol provided by manufacturer. Samples were treated with DNAse (ref. AM2224, AMbion) according to the manufacturer’s instructions and stored at −80 °C. RNA amount and quality was estimated using Qubit (Thermo Fisher Scientific) and the 2100 Bioanalyser instrument (Agilent). RNA libraries and sequencing were performed on the GenomEast platform, a member of the ‘France Génomique’ consortium (ANR-10-INBS-0009). Libraries were constructed using the TruSeq Stranded mRNA Library Prep Kit following the manufacturer’s recommendations. The libraries were sequenced on Illumina High HiSeq 4000 with paired-end 100 base pair long reads.
5
ChIP-seq Profiling of Hematopoietic Transcription Factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All ChIP experiments were performed after single crosslink with 1% methanol-free formaldehyde for 10 min, as reported (Obier et al., 2016 (link)). RUNX1, RUNX1-ETO (HA tag), LMO2, and LDB1 ChIP experiments (0 and 5 ng/ml Dox samples) were performed on CD34+ magnetic sorted progenitors. H3K4me3, H3K27ac (0 and 5 ng/ml Dox samples) and RUNX1 and RUNX1-ETO (HA tag) (0, 5 and 10 ng/ml Dox samples) were performed on non-adherent mixed progenitors (> 30% CD34+). Antibodies used are listed in Key Resources Table . ChIP libraries for Illumina sequencing were prepared using the KAPA Hyper Prep Kit, as detailed by the manufacturer. Quality control of the libraries was performed using a High Sensitivity DNA chip on an Agilent Technologies 2100 Bioanalyser instrument and libraries were quantified using a KAPA Library Quantification Kit. Libraries were sequenced in a pool of twelve indexed libraries in a NextSeq (Illumina) machine and a NextSeq® 500/550 High Output 75 cycle sequencing kit v2.
6
Microfluidic Separation and RISA-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Microfluidic separation was performed with a 2100 Bioanalyser Instrument (Agilent Technologies Ltd, Craven Arms, UK) using DNA 7500 chip (Agilent Technologies Ltd, Craven Arms, UK) [16] .
PCR bands of 55 different hospital samples were cluster-analysed with Bionumerics software (Applied Maths, Gent, Belgium) to determine the similarity of RISA-PCR profiles between hospitals and wards. Similarity was determined using Pearson coefficient [17] .
PCR bands of 55 different hospital samples were cluster-analysed with Bionumerics software (Applied Maths, Gent, Belgium) to determine the similarity of RISA-PCR profiles between hospitals and wards. Similarity was determined using Pearson coefficient [17] .
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!