RNA extraction and RT-qPCR were carried out as previously described28 (link). In brief, all strains were cultivated in liquid GMM supplemented with 5% yeast extract at 37 °C/250 rpm for 18 h. Mycelia were harvested, frozen in liquid nitrogen, and ground using a pestle and mortar. Total RNA was extracted using Qiagen RNeasy Mini Kit following the manufacturer’s protocol. DNA contamination from RNA samples was eliminated by RNase‐free Turbo DNase Kit (Invitrogen). Subsequently, cDNA was synthesized using SuperScript II system (Invitrogen), following the manufacturer’s instructions. Quantitative real-time PCR was carried out using SYBR® Green Master Mix (Bio‐Rad) in a CFX Connect Real‐Time System (Bio‐Rad).
Rnase free turbo dnase kit
Manufactured by Thermo Fisher Scientific
The RNase‐free Turbo DNase Kit is a laboratory product designed to effectively remove DNA from RNA samples. It utilizes a recombinant DNase enzyme that is engineered to be free of RNase activity, ensuring the preservation of the RNA during the DNA removal process. The kit provides a simple and efficient method for purifying RNA samples by eliminating contaminating DNA.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Sybr green master mix, by Bio-Rad (2 mentions)
Cfx connect real time system, by Bio-Rad (2 mentions)
Superscript 2 system, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Rneasy kit, by Agilent Technologies (2 mentions)
Agilent quick amp kit, by Agilent Technologies (1 mentions)
Quickamp kit, by Agilent Technologies (1 mentions)
4 protocols using rnase free turbo dnase kit
1
RNA Extraction and RT-qPCR Protocol
2
RNA Extraction and qPCR Analysis Protocol
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RNA extraction and RT-qPCR were carried out as previously described (22 (link)). In brief, all strains were cultivated in liquid GMM supplemented with 5% yeast extract at 37°C/250 rpm for 18 h. Mycelia were harvested, frozen in liquid nitrogen, and ground using a pestle and mortar. Total RNA was extracted using Qiagen RNeasy Mini Kit following the manufacturer’s protocol. DNA contamination from RNA samples was eliminated by RNase-free Turbo DNase Kit (Invitrogen). Subsequently, cDNA was synthesized using SuperScript II system (Invitrogen), following the manufacturer’s instructions. Quantitative real-time PCR was carried out using SYBR® Green Master Mix (Bio-Rad) in a CFX Connect Real-Time System (Bio-Rad).
3
RNA Extraction and Labeling for Microarray
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RNA was extracted from the cortex, hippocampus, hypothalamus and brainstem using Trizol (Invitrogen, Carlsbad, CA) and following the manufacturer’s directions. The RNA was resuspended in RNAsecure, and stored at -80°C in aliquots until use. For microarray analysis, 20 ug of RNA was DNase treated using the Turbo RNase-free DNase kit (Ambion, Foster City, CA), the concentration determined with a Nanodrop spectrophotometer (ND-1000, ThermoFisher, Wilmington DE) and the integrity of the RNA was measured using an Agilent Bioanalyzer, 2100 model. One μg of the DNase-treated RNA was labeled with Cyanine 3 (Cy3) CTP with the Agilent Quick Amp kit (5190–0442, New Castle, DE) according to their methodology, purified with the Qiagen RNeasy kit (Valencia, CA) as according to Agilent’s revision of the Qiagen protocol as shown in the Quick Amp kit protocol except that the microcentrifugation spins were performed at room temperature instead of 4°C. The resulting labeled cRNA was analyzed with the Nanodrop spectrophotometer, and the specific activities and the yields of the cRNAs were calculated. The labeled cRNA was stored at -80°C until use.
4
Hypothalamic RNA Extraction and Microarray Analysis
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RNA was extracted from the entire hypothalamus using Trizol (Invitrogen, Carlsbad, CA) following the manufacturer's directions. The RNA was resuspended in RNAsecure, and stored at −80°C in aliquots until use. For microarray analysis, 60 μg of these RNAs were DNase treated using the Turbo RNase‐free DNase kit (Ambion, Foster City, CA), the concentration determined with a Nanodrop spectrophotometer (ND‐1000, ThermoFisher, Wilmington, DE) and the integrity of the RNA was measured using an Agilent Bioanalyzer, 2100 model. The RNA Integrity Number (RIN) value for the RNAs ranged from 7.1 to 8.6. One microgram of the DNase‐treated RNA was labeled with Cyanine 3 (Cy3) CTP with the Agilent Quick Amp kit (5190‐0442, New Castle, DE) according to their methodology, purified with the Qiagen RNeasy kit (Valencia, CA) according to Agilent's revision of the Qiagen protocol as shown in the Quick Amp kit protocol except that the microcentrifugation spins were performed at room temperature instead of 4°C. The resulting labeled cRNA was analyzed with the NanoDrop spectrophotometer, and the specific activities and the yields of the cRNAs were calculated; these ranged from 10.22 to 12.38 pmol Cy3/μg RNA and from 5.6 to 8.9 μg, respectively. The labeled cRNA was stored at −80°C until use.
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