The CC was dissected from brains of experimental mice using the dissecting microscope. Three dissected CCs were pooled as one sample for total RNA isolation. Total RNA was extracted from these tissues using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocols. The RNA concentration was measured with a NanoDrop 1000 device (Thermo Fisher Scientific). Complementary DNA was synthesized using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Thermo Fisher Scientific). Real-time qPCR analysis was performed using the iTaq Universal SYBR Green Supermix (Bio-Rad). All primers were exon spanning (Sigma-Aldrich). Gene expression of C1ra, Cd209a, Cd36, Cnn1, H2-A2, H2-Eb1, Itgm, Nos1, Rac1, and Thbs4 was analyzed in the CC after 6 wk of CPZ treatment in Qk-iCKO mice and control mice. Information regarding the primers used is shown in Table S3 . The ΔΔCt method was used to determine differences in gene expression between Qk-iCKO mice and control mice. Changes in mRNA expression levels were calculated after normalization to β-actin.
Nanodrop 1000 device
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NanoDrop 1000 is a spectrophotometer designed for the accurate measurement of nucleic acid and protein concentrations. It utilizes a patented sample retention system that requires only 1-2 microliters of sample to perform a measurement. The device provides absorbance readings at various wavelengths to determine the concentration and purity of the sample.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Superscript 3 one step rt pcr system with platinum taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Itaq universal sybr green supermix, by Bio-Rad (1 mentions)
Random hexanucleotide primers, by Thermo Fisher Scientific (1 mentions)
Peqgold trifast, by Avantor (1 mentions)
Aceq qpcr sybr green master mix, by Vazyme (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Rna 6000 pico kit, by Agilent Technologies (1 mentions)
Nucleospin soil kit, by Macherey-Nagel (1 mentions)
Fastdna spin kit for soil, by MP Biomedicals (1 mentions)
Rq1 rnase free dnase, by Promega (1 mentions)
Invitrap spin rna mini kit, by Stratec (1 mentions)
Fastprep 24 device, by MP Biomedicals (1 mentions)
Lysing matrix d, by MP Biomedicals (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
7 protocols using nanodrop 1000 device
1
qPCR Analysis of CC in CPZ-Treated Mice
2
Gene Expression Analysis in Demyelination and EAE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gene expression studies were performed on corpus callosum tissue isolated from five healthy and five animals fed 0.25% cuprizone in the standard rodent chow for 3 weeks, followed by 17 days on normal chow, and on spinal cord tissue isolated from four healthy and three EAE mice on day 17 post EAE induction. Total RNA was extracted using peqGOLD TriFast (VWR), and RNA concentration and purity were measured with the NanoDrop 1000 device (Thermo Scientific). cDNA synthesis was performed using moloney murine leukemia virus reserve transcriptase kit and random hexanucleotide primers (Thermo Fisher Scientific). cDNA levels were then analyzed by RT‐rtPCR using AceQ® qPCR SYBR Green Master Mix (Vazyme). The expression levels were calculated relative to the reference gene coding for hypoxanthin‐guanin‐phosphoribosyl‐transferase using the ΔΔCt method. Primer sequences and individual annealing temperatures are shown in Table 1 .
3
Transcriptomic Response to GFS Feeding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The transcriptomic response upon GFS feeding was assessed using microarray analyses. Total RNA extraction and purification were performed on ground fresh leaf material using the RNeasy® Plant Mini kit (Qiagen) and DNAse I (Fermentas, RNAse-free) following the manufacturer’s instructions. Quality and quantity of the RNA were estimated using a NanoDrop 1000 device (Thermo Fisher Scientific, Waltham, MA, United States) and 1.5% agarose gel electrophoresis. Equal quantities of total RNA from four individuals of a single plant population at each time point were pooled, resulting in four total RNA samples per treatment (control or induced). The RNA concentration, integrity and purity were assessed by electrophoretic analysis with the RNA 6000 Pico Kit using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, United States)1 . All samples had an RNA integrity number (RIN) between 6.6 and 7.2 and were hybridized on 8x60K Agilent microarrays. cDNA labeling, microarray hybridization, design were performed as described in Lortzing et al. (2017) (link). The design and the experimental data of the microarray are available at the Gene Expression Omnibus of the National Centre for Biotechnology Information (NCBI; GEO Accession: GSE131208 ). RT-qPCR analyses were performed for technical validation of the microarrays (see Supplementary Data Sheet S1 and “Statistical Analyses” section).
4
Hippocampal Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Microarray data from our previous study [26 (link)] using the hippocampus of 12- and 52-week-old mice were re-analyzed. The microarray data were deposited into the GEO database (accession number GSE144459). Total RNA was isolated from the frozen hippocampus using the RNeasy Mini Kit (QIAGEN, #74104). A NanoDrop1000 device (Thermo Fisher Scientific) was used to measure the concentrations of RNA. An Agilent 2100 Bioanalyzer (Agilent) was used to measure the RNA integrity number (RIN). All of the samples used for the microarray analysis satisfied the following criteria: A260/A280≥1.8, A260/A230≥2.0, and RIN≥7.0. The RIN of each sample is shown in Supplementary Table 1 .
5
Soil DNA and RNA Extraction Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was extract from approximately 500 mg of each soil sample using the Fast DNA Spin Kit for Soil (MP Biomedicals, USA). The DNA was purified using the NucleoSpin Soil Kit (Macherey-Nagel, Germany) from the sixth step of its extraction protocol, due to residual presence of humic acids after the final step of extraction.
RNA was extracted from approximately 2 g of soil using the RNA Power Soil – Total RNA Isolation Kit (Mobio, USA) according to the manufacturer’s protocol. After the RNA extraction, 7 μL were treated with RQ1 RNase-Free DNase (Promega, USA) to remove any DNA contamination.
Nucleic acid purity and concentration was assessed using a NanoDrop 1000 device (Thermo Fisher Scientific, USA) and a Qubit 3.0 fluorometer (Thermo Fisher Scientific, USA), respectively.
RNA was extracted from approximately 2 g of soil using the RNA Power Soil – Total RNA Isolation Kit (Mobio, USA) according to the manufacturer’s protocol. After the RNA extraction, 7 μL were treated with RQ1 RNase-Free DNase (Promega, USA) to remove any DNA contamination.
Nucleic acid purity and concentration was assessed using a NanoDrop 1000 device (Thermo Fisher Scientific, USA) and a Qubit 3.0 fluorometer (Thermo Fisher Scientific, USA), respectively.
6
Quantifying 8-oxodGuo in Knockout Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Snap-frozen brain, heart, lungs, liver and kidneys were stored at -80 °C until DNA extraction.
These were processed for 8-oxodGuo determination in all of the MTH1 knock-out mice and a selection of the XPA, XPC and CSB animals.
Extraction of DNA from 90-150 mg of homogenised tissue (outer layer removed with a scalpel; homogenised in a hand-held homogeniser) was performed according to a protocol recommended by the European Standard Committee on Oxidative DNA Damage (ESCODD) designed to minimise adventitious formation of 8-oxodGuo [31] . Extracted DNA was dissolved in nuclease-free water overnight at room temperature on a rotating mixer, then quantified by a Nanodrop 1000 device (ThermoFisher Scientific, Loughborough, UK). A volume equivalent to 50 µg DNA was dried in a SpeedVac, reconstituted in DNA digestion buffer, enzymatically digested and analysed by HPLC-MS/MS according to the protocol reported by Singh et al. [32] . Heart tissues consistently yielded low amounts of DNA and were not analysed further.
These were processed for 8-oxodGuo determination in all of the MTH1 knock-out mice and a selection of the XPA, XPC and CSB animals.
Extraction of DNA from 90-150 mg of homogenised tissue (outer layer removed with a scalpel; homogenised in a hand-held homogeniser) was performed according to a protocol recommended by the European Standard Committee on Oxidative DNA Damage (ESCODD) designed to minimise adventitious formation of 8-oxodGuo [31] . Extracted DNA was dissolved in nuclease-free water overnight at room temperature on a rotating mixer, then quantified by a Nanodrop 1000 device (ThermoFisher Scientific, Loughborough, UK). A volume equivalent to 50 µg DNA was dried in a SpeedVac, reconstituted in DNA digestion buffer, enzymatically digested and analysed by HPLC-MS/MS according to the protocol reported by Singh et al. [32] . Heart tissues consistently yielded low amounts of DNA and were not analysed further.
7
Testicular RNA Extraction and Transcriptional Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Testicular RNA was extracted of 12-to 13-week-old males. For the transcriptional analysis, we used 16 mice per line, subdivided into two separate sets of biological replicates (each of eight animals per set and line). Mice were killed by CO 2 inhalation, and the testes were dissected to be either snap frozen in liquid N 2 or preserved in RNAlater (Ambion). Tissue disruption was done in RLT buffer (RNeasy kit, Qiagen) using a FastPrep-24 device (MP Biomedicals, Santa Ana, CA, USA) and Lysing Matrix D disposable tubes. RNA was extracted by the phenol/chloroform method, followed by spin column chromatography using the RNeasy kit (Qiagen) protocol according to the manufacturer's instructions.
In the case of snap-frozen tissue the InviTrap Spin RNA Mini Kit (Stratec, Birkenfeld, Germany) was used for whole RNA extraction. The quantification was done spectrophotometrically using a NanoDrop 1000 device (Thermo Fisher Scientific), and the RNA integrity was checked by capillary electrophoresis (Agilent 2100 Bioanalyzer).
In the case of snap-frozen tissue the InviTrap Spin RNA Mini Kit (Stratec, Birkenfeld, Germany) was used for whole RNA extraction. The quantification was done spectrophotometrically using a NanoDrop 1000 device (Thermo Fisher Scientific), and the RNA integrity was checked by capillary electrophoresis (Agilent 2100 Bioanalyzer).
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!