illustra™ RNAspin Mini Isolation Kit

Protocol tips

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- This kit can be used for the isolation of RNA from up to 5x10^6 cells

Upstream tips
- This kit can be used for the isolation of RNA from up to 5x10^6 cells

Publication protocol

The cells were cultured in 24-well plates at a concentration of 2 × 106 cells/well in a 5% CO2 atmosphere at 37 °C. After adhesion, the cells were treated for 6 h with the chemicals at a concentration that permitted ~70% cell viability. Next, mRNA was extracted from the cells and purified using the Illustra RNAspin Mini RNA Isolation kit (GE Healthcare). After treatment with DNAse I (Sigma-Aldrich), 1 µg mRNA was used for synthesis of cDNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). Messenger RNA was quantified in a total volume of 20 μL using the TaqMan® Gene Expression Assay (Applied Biosystems) according to manufacturer instructions. The reaction mixture contained 1 µL 20X TaqMan® Gene Expression Assay (TaqMan® Gene Expression Assay IDs are shown in Table 1), 10 µL 2X TaqMan® Gene Expression Master Mix, 1 µL cDNA, and 8 µL RNase-free water. A negative control was included for each gene and amplification reactions were performed in triplicate in an Mx3005P real-time thermocycler (Stratagene, La Jolla, CA, USA), with one hold at 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The fluorescence data were collected at the end of the annealing/elongation step. Data were analyzed and plotted using the MxPro software (Stratagene). The RPL32 gene was used as a reference for normalization of the results and fold-differences in gene expression were calculated relative to control samples (cells treated with 0.5% DMSO) using the ΔΔCT method.

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Manufacturer protocol

Download the product protocol from GE Healthcare Life Sciences for illustra™ RNAspin Mini Isolation Kit below.

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