We have described a novel method and instrument, RCC, as a new platform technology for RNA isolation. Isolation of all species of E. coli
tRNAs and various ncRNAs from yeast and mouse demonstrate that RCC is a powerful, efficient, reproducible and convenient method for isolating multiple species of RNA molecules from a single sample of total RNA. To establish this method as a generally applicable method for RNA research, it will be necessary to optimize and improve some aspects of this method. As shown in this study, RNA molecules were isolated with various purities and yields. These parameters depend on several factors, including probe design, temperature control and salt concentration. For isolating stable RNA molecules like tRNAs, probe design is the most important factor to achieve maximum purity and yield. Chain length, GC content and sequence specificity must also be considered for each DNA probe. For 44 E. coli
tRNAs, we designed 30 mer DNA probes complementary to the anticodon or 3′ region of each tRNA, depending on the sequence specificity. To optimize design of probes for each target, northern or dot blots are convenient for checking the hybridization efficiency of each probe (28
). RCC might also be used to compare the efficiency and performance of several different probes for a single target RNA.
According to our theoretical model for RCC, the total yield of target RNA was closely correlated to the equilibrium constant (K
) of ligand–target interaction. For efficient isolation of RNA molecules, it might be better to choose nucleic-acid-related compounds as probes (30
), such as the locked nucleic acid (LNA) or the peptide nucleic acid (PNA), which have higher hybridization efficiencies than a DNA probe. Since a small fraction of biotinylated DNA probes detached from the streptavidin sepharose during the reciprocal circulating step, further investigation of resins and methods for immobilization of probes is needed. DNA probes having an amino-linker covalently bound to sepharose resin activated with N
-hydroxysuccinimidyl ester may be a suitable alternative choice for immobilization. Furthermore, the temperature for hybridization and the buffer composition for each purification step is largely dependent on the particular probe. Since RCC basically has to use a single condition for 8 different probes in one operation, it is important to find conditions which are suitable for all the probes being used. Theoretical predictions of target RNA yield derived from a simplified model of the RCC method were closely correlated with the experimental results. The required number of cycles and yields of individual RNA species can be predicted by this model if the value of K
is known. The theoretical model is useful to optimize the conditions and operation of the RCC instrument.
Preparation of total RNA is another critical factor for isolation of RNA species. Highly concentrated total RNA provides the best starting material, especially for isolation of minor RNA species. However, highly concentrated total RNA is often very viscous due to polysaccharides and other cellular components which are co-extracted from the cell. Prior to RCC, an initial separation of total RNA by anion exchange chromatography is required to reduce the viscosity of the concentrated total RNA solution.
RCC could easily be scaled up for high throughput applications. The number of channels in the RCC prototype instrument we assembled could be varied from one to eight, but a 96- or 384-channel liquid handling instrument could easily be used for RCC. In such cases, the RCC instrument could be used not only as a high-throughput purification machine, but also as an analytical device for various applications in RNA research. In addition, since the sample volume can be changed, minor RNA species can be purified from large volumes of an initial RNA sample, and ncRNAs such as miRNAs or piRNAs are good targets to be isolated by this method. Most of the miRNAs are regulatively expressed in specific physiological conditions, such as pathological cells, tissues and organs. It is impossible to isolate each miRNA from limited quantity of specimens. To profile miRNA expression pattern routinely, high sensitive detection systems such as microarray analysis or RT-PCR are now available. However, qualitative aspects of miRNAs are also important, because it is known that miRNAs have several dicing variants with different length and variable termini, which might modulate their activity and change its target specificity. To investigate such qualitative information, it will be of importance to isolate each miRNA once at least (not routinely) from relatively large amount of specimen. Mass spectrometry analysis of the purified RNAs can precisely quantify processing variants and/or identify the chemical structure of RNA modifications if present.
The RCC method can be used in a variety of applications. For example, when antibodies are immobilized on the tip-columns, RCC could be used for automated multiple-immunoprecipitations for interactome analysis. RCC has great potential to be used in a wide variety of applications requiring isolation of multiple components. Continued refinement of the operating conditions and testing of additional applications will validate the potential of RCC.