Researchers performing array analysis have found that a combination of phenol based and solid phase RNA extraction methods often provide superior array data than either method alone (1-3). Ambion's new line of RiboPure RNA Isolation Kits are the first kits that combine these RNA extraction methods. Four different kits are now available for the isolation of high quality total RNA from a variety of samples:
RiboPure Kit for isolating RNA from animal and plant tissues and cells
RiboPure-Yeast Kit for isolating RNA from yeast cells
RiboPure-Bacteria Kit for isolating RNA from microbes
RiboPure-WBC Kit for RNA isolation from blood samples
High Quality RNA Isolation
The RiboPure RNA Isolation Kits combine a single reagent homogenization and organic extraction step with traditional glass fiber filter purification to yield exceptionally pure RNA, free of residual proteins and lipids. The RiboPure procedure is especially suited for processing problematic samples that would otherwise clog filter cartridges (e.g. muscle, heart), samples that are difficult to disrupt (e.g. muscle, fibrous plant tissue, yeast, bacteria), or samples that are high in ribonuclease or other contaminants (e.g. spleen, pancreas, plant and neural tissues). RiboPure is the method of choice for preparing RNA for use in microarray analysis or other procedures that require RNA of exceptional purity. Each of the RiboPure Kits described below are based on this dual method technology and also include Ambion's DNA-free™
genomic DNA removal system, making them ideal for preparation of RNA for RT-PCR based applications.
Fast Results and High Yields with Small Samples
The RiboPure Kit employs a rapid, 15 minute RNA purification procedure that combines the best features of one step organic extraction protocols and solid phase extraction techniques. From 1 to 100 mg of tissue or 100 to 107 cells can be processed per purification. Briefly, the protocol consists of disrupting samples in Ambion's RNAwiz™, which contains phenol and RNase inhibitors, then adding chloroform and mixing. The sample is then centrifuged to separate organic and aqueous phases. The aqueous phase, which contains the RNA, is recovered and mixed with ethanol. This preparation is then passed over an RNA-binding silica filter. The filter is washed to remove contaminants, and the RNA is eluted in a small volume. A subsequent DNA removal step adds ~30 minutes of additional time (mostly incubation time) to the procedure. Total RNA yield is typically 80-400 µg per 100 mg tissue, depending on the type of tissue processed. The RiboPure Kit contains reagents for up to 50 RNA isolations.
New! Isolate High Quality Total RNA from Yeast
In yeast, gene expression patterns can be affected during pretreatments used to disrupt the yeast cells prior to RNA isolation. The RiboPure-Yeast RNA Isolation Kit uses no enzymatic pretreatments and thus does not alter expression profiles. RiboPure-Yeast combines a simple and efficient glass bead/one step phenol mediated disruption using Ambion's Vortex Adapter
followed by a glass filter based RNA purification step. After DNase treatment with the included DNA-free™ Reagents, the resulting RNA is exceptionally pure (figure 1) containing little to no genomic DNA (<0.05%). The kit has been used at Ambion to isolate high quality RNA from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, and Candida albicans. The kit provides the highest yield of any method tested from each of these species. The RiboPure-Yeast RNA Isolation Kit contains sufficient reagents for 50 RNA isolations each from 107
up to 3 x 108
Figure 1. Yeast Total RNA Isolated Using the RiboPure-Yeast Kit.
P. pastoris, S. cerevisiae RNA, C. albicans, and S. pombe RNA, isolated using the RiboPure-Yeast Kit and treated with DNase, was run on an Agilent bioanalyzer chip. The ratio of 28S:18S rRNA is given for each sample.
Isolate High Quality Total RNA from Bacteria
As with yeast, bacterial gene expression patterns can also be affected during enzymatic or chemical pretreatments used to disrupt cell walls prior to RNA isolation. The RiboPure-Bacteria RNA Isolation Kit uses no enzymatic pretreatments, allowing for faster processing, which preserves RNA expression profiles. The kit utilizes the same type of protocol combining an efficient glass bead/one step phenol mediated disruption with the Vortex Adapter followed by a glass filter based RNA purification step. The kit has been used at Ambion to isolate high quality RNA from Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Campylobacter fetus, and Rhodobacter sphaeroides and is compatible with most bacterial RNA species (Figure 3). The RiboPure-Bacteria RNA Isolation Kit contains sufficient reagents for 50 RNA isolations, each from 107
up to 109
Figure 3. High Quality Bacterial RNA Obtained with RiboPure-Bacteria Kit.
Total RNA (2 µg) was separated on a 1% agarose gel in formaldehyde/MOPS buffer. Samples were heated at 80ºC for 10 min in denaturing agarose gel loading buffer with 100 µg/µl EtBr before electrophoresis at 5 V/cm
RiboPure-WBC: Scalable Protocol for Isolating High Quality RNA from Blood
Isolating intact RNA from blood is challenging because: 1) only white blood cells (WBCs) contain appreciable amounts of RNA and WBCs make up only about 0.2% of blood cells and 2) whole blood also contains large amounts of proteins, heme, and genomic DNA, as well as intracellular and extracellular RNases. Thus, WBCs typically need to be isolated away from the rest of the blood constituents prior to RNA isolation.
The RiboPure-WBC Kit protocol consists of three steps: 1) fractionation of whole blood by centrifugation, recovery of the WBCs and RNA stabilization in Ambion's RNAlater®
(provided), 2) lysis of WBCs in guanidinium and initial RNA purification by phenol/chloroform extraction, and 3) final RNA purification on a glass fiber filter.
The kit provides sufficient reagents for the purification and stabilization of WBCs from 40 whole blood samples of up to 10 ml each (2.5-10 ml of whole blood can be used per isolation). The RNA purified with the RiboPure-WBC Kit can be used in the most demanding downstream applications, such as real-time RT-PCR or array analysis. Average yields of RNA range between 4-20 µg from 5 ml of whole blood; the variation is due to differences in WBC numbers between samples.