RNA isolation and microarray hybridization were performed as described previously [59 (link)]. In brief, RNA for microarray analysis was extracted using TRIzol reagent (Invitrogen) and purified using TRIzol® Plus RNA Purification Kit (Sigma-Aldrich) according to the instructions of the manufacturer. The concentration of RNA was calculated from the absorbance at 260 nm in a spectrophotometer (Biochrom Libra S22). The quality of the RNA was analyzed with an Agilent 2100 Bioanalyzer using a RNA6000 LabChip kit (Agilent Technology). Microarray hybridization using the Affymetrix GeneChips A. niger Genome Array was performed at GenomeScan (Leiden, The Netherlands).
Trizol plus rna purification kit
Manufactured by Merck Group
The TRIzol® Plus RNA Purification Kit is a reagent designed for the isolation and purification of total RNA from various biological samples. It uses a guanidinium thiocyanate-phenol-chloroform extraction method to effectively separate and isolate RNA from DNA, proteins, and other cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Libra s22, by Harvard Bioscience (2 mentions)
Rna 6000 labchip kit, by Agilent Technologies (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Agilent 2100, by Agilent Technologies (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
3 protocols using trizol plus rna purification kit
1
RNA Isolation and Microarray Analysis of Aspergillus niger
2
Transcriptome Analysis of Sugar Stress
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Samples from 12, 18, 24 and 33 h were used for microarrays to evaluate the influence of the changes in extracellular sugar concentration on whole genome gene expression. These time points were chosen as they reflect the stage in which specific sugars were taken up. RNA was extracted using TRIzol reagent (Invitrogen) and purified using TRIzol® Plus RNA Purification Kit (Sigma-Aldrich) according to the instructions of the manufacturer. The RNA concentration was calculated from the absorbance at 260 nm in a spectrophotometer (Biochrom Libra S22). The RNA quality was analyzed with an Agilent 2100 bioanalyzer using a RNA6000 LabChip kit (Agilent Technology). Microarray hybridization was performed at GenomeScan (Leiden, The Netherlands). Microarray data was analyzed using Affymetrix Expression Console software v1.4.1.46 with the normalized values in linear scale. The probe intensities were normalized for background by the robust multiarray average (RMA) method which makes use of only the perfect match probes. Gene expression values were calculated with the medianpolish summary method from the PM probes. Genome scale PCA analysis with gene expression of the four different time points was generated using FactoMineR34 (link) package from Rcomander v.2.1–7 in R 3.1.2.35 .
3
Total RNA Isolation and cDNA Synthesis from PBMCs
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Total RNA from PBMC cells was isolates with the TRIzolPlus-RNA purification kit (Sigma).
Using agarose gel electrophoresis, the RNA quality was determined. DNA residual in the extracted total RNA was removed by DNase1 treatment.
The quantity of RNA isolated was measured by NanoDrop Spectrophotometer (Thermo Scientific, USA).
Complementary DNA was synthesized from isolated RNA using RevertAid first strand cDNA synthesis kit (Thermo Scientific, K1622).
The reactions were set up in 0.2 ml PCR tubes.
Template RNA 5 μg, Oligo (dT) 18 primer 1.0 µl, 5X reaction buffer 4.0 µl, Ribolock 1 µl, dNTP mix (10 mM) 2.0 µl, Revertaid 1.0 µl and Nuclease free water. The contents of the tube were mixed gently and centrifuged briefly. For qRT PCR works, oligo (dT) 18 primer were used for cDNA synthesis, the reaction mix was incubated for 60 minutes at 42 o C.
The reaction was terminated at 70 o C for 5 minutes.
Using agarose gel electrophoresis, the RNA quality was determined. DNA residual in the extracted total RNA was removed by DNase1 treatment.
The quantity of RNA isolated was measured by NanoDrop Spectrophotometer (Thermo Scientific, USA).
Complementary DNA was synthesized from isolated RNA using RevertAid first strand cDNA synthesis kit (Thermo Scientific, K1622).
The reactions were set up in 0.2 ml PCR tubes.
Template RNA 5 μg, Oligo (dT) 18 primer 1.0 µl, 5X reaction buffer 4.0 µl, Ribolock 1 µl, dNTP mix (10 mM) 2.0 µl, Revertaid 1.0 µl and Nuclease free water. The contents of the tube were mixed gently and centrifuged briefly. For qRT PCR works, oligo (dT) 18 primer were used for cDNA synthesis, the reaction mix was incubated for 60 minutes at 42 o C.
The reaction was terminated at 70 o C for 5 minutes.
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