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RiboPure™ Total RNA Isolation Kit
Better RNA Isolation for the Most Stringent Downstream Applications
The improved RiboPure™ RNA Isolation Kit combines TRI Reagent® Solution, a robust lysis and denaturing reagent, with a glass-fiber filter purification method to provide RNA of high yield and quality. TRI Reagent Solution is a monophasic solution containing phenol and guanidine thiocyanate that reliably and rapidly lyses cells and simultaneously inactivates nucleases. The use of glass-fiber filters for RNA purification after phase separation with TRI Reagent Solution simplifies the process and eliminates impurities. Exceptionally pure RNA, free of proteins, organic solvents, and other contaminants, is obtained. The RNA also contains minimal genomic DNA contamination and is ideal for various downstream applications, including RNA amplification for microarray analysis, real-time RT-PCR, and Northern blotting.
Improved RiboPure RNA Isolation Protocol
The RiboPure RNA Isolation Kit is suitable for isolation of RNA from a broad range of biological samples, including 5-100 mg fresh, frozen, and RNAlater® Solution-treated tissues, and 0.1-20 million mammalian cultured cells. The protocol is fast, simple, reliable, and can be completed in less than 30 minutes. Biological samples are lysed and homogenized in TRI Reagent Solution and then extracted with bromochloropropane to remove cellular components such as proteins, lipids, and DNA. RNA in the aqueous phase is purified by binding to a glass-fiber filter, washing twice to remove residual contaminants (e.g., salt and organic solvents), and eluting from the filter using a low salt buffer.
Successful Microarray Experiments with High Quality RNA
Obtaining high quality total RNA is critical for successful microarray experiments that accurately capture the expression profile of a biological sample. Using total RNA from RNAlater-treated mouse lung, the RiboPure method was compared to the Affymetrix® recommended protocol, which also involves phenol-based sample lysis followed by glass-fiber filter RNA purification (Figure 1). Comparable RNA yield and quality were obtained from both protocols. Excellent correlation exists between normalized microarray signal intensities obtained from the two total RNA samples isolated using the RiboPure Kit (Figure 2A). Excellent correlation was also observed between total RNA samples purified using RiboPure and the Affymetrix recommended protocol (Figure 2B). High percent Present calls (>60%) were obtained for total RNA purified using either procedure, but approximately 2% higher Present calls (~454 genes) were observed in the RiboPure purified samples (Figure 2C). The results of these experiments indicate the suitability of the RiboPure RNA Isolation Kit for the most stringent downstream applications.
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| Figure 1. High Yield and Quality RNA Isolated from RNAlater® Solution-treated Mouse Lung Using the RiboPure™ Total RNA Isolation Kit. RNAlater solution-treated mouse lung (100 mg) was homogenized in TRI Reagent® Solution (1 mL). RNA isolation was performed in duplicate using 1 mL aliquots of tissue homogenate using the RiboPure and Affymetrix® Recommended Protocols. Yield and A260/A280 were determined using a NanoDrop® Spectrophotometer, and 28S/18S rRNA ratios and RIN values were determined using an Agilent® 2100 bioanalyzer. |
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| Figure 2. High Quality Microarray Data Using Total RNA Isolated with the RiboPure™ Kit. Total RNA samples described in Figure 1 (1 µg each) were amplified and biotinylated using the MessageAmp™ II-96 aRNA Amplification Kit. Each biotin-labeled, amplified RNA sample was fragmented and hybridized to an Affymetrix® GeneChip® Mouse Genome 430A 2.0 Array (total probe set=22,000) and analyzed with the Affymetrix GeneChip Operating Software. (A) Scatter plot of normalized signal intensities from microarray experiments using RiboPure Kit-purified total RNA; correlation between the two samples is 0.997. (B) Scatter plot of normalized signal intensities from microarray experiments using total RNA purified using the RiboPure Kit and Affymetrix recommended protocol; correlation between the two methods is 0.995. (C) Greater than 60% Present calls were obtained for all samples; ~2% higher percent Present calls (~454 genes) were observed using the RiboPure Kit. |
Scientific Contributors
WeiWei Xu, Quoc Hoang, Roy Chris Willis, Angela Burrell, Xiaowei Wang, Mangkey Bounpheng, Xingwang Fang • Applied Biosystems, Austin, TX |
