RNeasy Plus Mini Kit

Protocol tips

Upstream tips	Protocol tips	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

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

Publication protocol

Total RNA was extracted from the sixteen cell lines harvested at log phase, using the RNeasy® Plus (Qiagen) kit following the manufacturer’s protocol. For real-time quantitative PCR (qPCR), mRNA was reverse-transcribed into cDNA using an ABI RT kit (Applied Biosystems). qPCR primers and probes were designed using Roche Probe Finder Design Software (Roche Applied Sciences) and reactions performed using the ABI PRISM 7900 Sequence Detection System instrument and software (Applied Biosystems). The reactions were incubated in a 384-well optical plate at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 10 min. Experiments were performed in triplicate for each sample. Gene expression was normalized to internal control SDHA (succinate dehydrogenase), YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide).

Cells were collected from 226L-U19, HB4a, MCF10a, MCF7, T47D and SKBR3 cell lines cultured in monolayer (pre-incubated with MS media for 12 hrs) and from viable AR cells. RNA was extracted using the RNeasy® Plus Mini (Qiagen) following the manufacturers protocol. In addition to the gDNA eliminator spin column provided in the RNeasy kit, an on-column DNase digestion was performed (RNase-Free DNase Set; Qiagen) to ensure maximum removal of DNA. RNA was eluted and the concentration measured using a GeneQuant machine (Amersham Biosciences). RNA integrity was assessed by microanalysis (Agilent Bioanalyser) and amplified using the WT-Ovation™ Pico RNA Amplification System (NuGEN) following the manufacturers protocol which employs SPI™ amplification (linear isothermal DNA amplification process) to amplify the RNA about 15,000-fold.

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Manufacturer protocol

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

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