RNeasy Mini Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).	- Include DNAse treatment for 15-20min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C. - For purifying RNA add either 10 μl β-mercapto ethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT).

Protocol tips
- To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).
Downstream tips
- Include DNAse treatment for 15-20min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C. - For purifying RNA add either 10 μl β-mercapto ethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT).

Publication protocol

Total RNA was extracted from IFN-γ– or LPS-treated or untreated cells with the RNeasy Mini Kit (catalog number 74104; Qiagen) according to the manufacturer's instructions. Two micrograms of total RNA was used for cDNA synthesis, using SuperScript II reverse transcriptase (catalog number 18080-044) and random primers (catalog number 48190-011; Invitrogen) in a 20-μl reaction system, with the following cycles: 25 °C for 5 min, 42 °C for 50 min, and 75 °C for 5 min. After the reaction was complete, the RT-PCR products were diluted with diethylpyrocarbonate/H2O to 100 μl for real-time PCR. Real-time RT-PCR was performed on 384-well plates in a 20 μl reaction system containing 2 μl of diluted cDNA, 1 μl of primer mixture, 7 μl of H2O, and 10 μl of TaqMan 2× reaction mixture. PCR was carried out under default cycling conditions, and fluorescence was detected with the ABI 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). Triplicate determinations were performed for each sample that was used for real-time PCR; the mean value was calculated, and the data in the final figures represent the results of three independent experiments. Relative gene expression was calculated as the ratio of the target gene to the internal reference gene (β-actin) multiplied by 106 based on Ct values.

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Manufacturer protocol

Download the product protocol from Qiagen for RNeasy Mini Kit below.

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