Total RNA and DNA were isolated using Tri-Reagent extraction (Sigma–Aldrich, Munich, Germany) starting from 50–100 mg of tissues (depending on the initial specimen size). RNA quality and yield were verified by Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) using a RNA 6000 Nano Kit (Agilent Technologies, Ltd.) and NanoDrop 2000 (Thermo Scientific, NanoDrop products, Wilmington, USA). DNA purity was estimated using NanoDrop 2000 (Thermo Scientific, NanoDrop products, Wilmington, USA).
Tri reagent extraction
Manufactured by Merck Group
Sourced in Germany
Tri-Reagent extraction is a laboratory technique used for the isolation and purification of RNA, DNA, and proteins from a variety of biological samples. The core function of this product is to provide a single-step method for the extraction and separation of these biomolecules from cells or tissues.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Rna 6000 nano kit, by Agilent Technologies (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Avantor (1 mentions)
5 protocols using tri reagent extraction
1
Total RNA and DNA Extraction
2
RNA Extraction Using TriReagent Protocol
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RNA was extracted by using a modified version of a previously described procedure [73 (link)]. Total RNA from cell cultures was prepared by TriReagent extraction procedure (#T9424; Sigma-Aldrich), following manufacturer’s instructions. Briefly, the cells were lysed in the appropriate volume of cold Tri-Reagent for 5 min and separated in three phases by adding chilled chloroform and by centrifuging at 12,000 g. The upper aqueous phase was transferred to a clear pre-chilled tube and the RNA was precipitated for 40 min, in cold isopropyl alcohol, and then centrifugated at 12,000 g. The obtained RNA pellets were washed 2 times with ice-cold 75% ethanol and upon centrifugation at 7500 g resuspended in 50 µL of RNase-free water. The RNA yield was determined with NanoDrop2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
3
Robust RNA Extraction from Frozen Tissues
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Total RNA of frozen individual tissue samples was isolated with Tri-Reagent-extraction (Sigma-Aldrich, Taufkirchen, Germany) according to manufacturer’s protocol. DNase treatment and a column-based purification using the RNeasy Mini Kit (Qiagen, Hilden, Germany) were also performed according to manufacturers’ protocols. To check RNA integrity, samples were visualized on 1 % agarose gels containing ethidium bromide. RNA concentration was determined by spectrometry with a NanoDrop ND-1000 spectrophotometer (PEQLAB, Erlangen, Germany). The absence of DNA contamination was confirmed by using the RNA as a template in standard PCR to amplify fragments of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. To prevent degradation, all RNAs were stored at −80 °C until further use.
4
Total RNA Isolation from Tissue
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Total RNA of individual tissue samples (n = 88) was isolated by Tri-Reagent-extraction (Sigma-Aldrich, Taufkirchen, Germany). DNase treatment and column-based purification using the RNeasy Mini Kit (Qiagen, Hilden, Germany) were used to ensure purity. Quality of RNA was checked using 1% agarose gels containing ethidium bromide. Concentration of RNA was also detected by spectrometry with a NanoDrop ND-1000 spectrophotometer (PEQLAB, Erlangen, Germany). Additionally, the absence of DNA contamination was verified by using the RNA as template in standard PCR amplifying fragments of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. All RNAs were stored at −80°C until further use.
5
Differential Gene Expression in GBM and JPA
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Snap-frozen 13 GBM and 11 JPA were obtained from the Canadian Brain Tumor Tissue Bank (London Health Sciences Centre, Ontario CA) and the Children's Memorial Health Institute (Warsaw, Poland). Total RNA was prepared using Tri-Reagent extraction (Sigma-Aldrich, Munich, Germany) followed by the RNeasy Mini Kit (Qiagen, Hilden, Germany) isolation as described [23 (link)]. RNA quality/yield was verified by Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) and NanoDrop 2000 (Thermo Scientific, München Germany). Samples were analyzed on Human HT-12 Expression BeadChips at the Cambridge Genomic Core Facility, UK. Array data processing and analysis were performed using IlluminaGenomeStudio software and normalized using the R Bioconductor beadarray and limma packages as described. Probes not detected (P value > 0.01) on the microarrays were removed from further analysis.
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