As the genomics revolution moves from gene discovery to gene function analysis, techniques requiring RNA manipulation are becoming more prominent. Development of antisense RNA (aRNA) amplification technology has revolutionized gene expression analysis by allowing experimentation from minute amounts of starting RNA materials. Another technical breakthrough is RNAi, which is rapidly becoming an important tool for manipulating gene expression. The use of RNAi treatment coupled with detailed microarray studies based on aRNA amplification is a powerful combination of techniques useful for understanding gene expression and functional data. Procuring this type of information will be aided greatly by a high throughput method for purifying RNA or cDNA from the in vitro reactions associated with microarray and RNAi studies, namely cDNA synthesis, in vitro transcription, aRNA amplification and cDNA/RNA labeling.
Purification of RNA and cDNA from in vitro reactions has traditionally been performed usi ng precipitation or column-based reagents. LiCl preferentially precipitates RNA (Barlow et al.,1963), but requires a series of manual steps including chilling, washing and multiple centrifugations. LiCl precipitation has also been linked to decreased performance in downstream in vitro translation reactions possibly due to inhibition by chloride ions (Maniatis et al., 1989). Column purification does not require chilling, but it does require multiple centrifugation steps which make automation more challenging.
Solid Phase Reversible Immobilization (SPRI) is a patented magnetic bead-based purification technology (Hawkins et al., 1995). Nucleic acids are immobilized onto magnetic beads allowing for easy contaminant removal and sample washing. Purified nucleic acids are easily eluted in aqueous solutions. Optimization of SPRI immobilization buffers allows for selective isolation of specific nucleic acid species. Importantly, SPRI requires no centrifugation or filtration steps which enables simplified automation.
RNAClean purification of RNA or cDNA is faster, easier and more efficient than traditional methods such as LiCl precipitation. We compared purification of RNA ladders containing a wide range of fragment sizes (0.16 - 9.5kb) using LiCl or RNAClean. RNAClean had a higher percent recovery than standard LiCl precipitation for the entire range of RNA sizes (Figure 1, Table 1). Importantly, the smaller RNA fragments purified using RNAClean maintained the same proportional representation as in the unpurified samples.
Recovery at Low Concentrations
RNAClean is a versatile reagent that provides efficient recovery from a range of starting RNA concentrations. This can be important as the yield of in vitro produced RNA requiring purification can vary widely from application to application. We produced a 1.9kb RNA fragment through in vitro transcription and purified it using RNAClean (Figure 2). The concentration of the RNA transcript prior to purification ranged from over 100ng/l to 2 ng/l. RNAClean shows nearly 100% recovery from reactions containing RNA at concentrations as low as 12ng/l and over 60% recovery from starting RNA concentrations as low as 1ng/l.
Low Volume Elution
RNAClean allows elution in small volumes, which can serve as an effective means to concentrate the cDNA or RNA for downstream application flexibility. We purified a 1.9kb transcript using RNAClean with varied elution volumes from 10 to 40l of water (Figure 3). The percent recovery was greater than 80% for all elution volumes tested.
RNAClean-purified RNA is free of nucleases and other contaminates which could effect performance in downstream applications. Overall quality of RNA purified with RNAClean from an in vitro transcription reaction was assessed using the Agilent BioAnalyzer 2100. Before testing, the RNA was incubated at either 4C for 2 days, at room temperature for 2 days, or 37C for 2 hours. No detectable degradation was observed in any of the samples tested (Figure 4).
We also tested RNAClean-purified RNA for contamination with impurities that inhibit downstream reactions such as reverse transcription and PCR. RNA transcripts purified with LiCl precipitation or RNAClean were used as templates for first strand cDNA sy nthesis. The first strand products were subsequently used as templates for PCR amplification (Figure 5). RNAClean-purified nucleic acids were of high quality and showed no signs of inhibitor impurities.
RNA Amplification Reactions
Amplification of RNA is a powerful technique that enables probe preparation for microarray analysis from very minute amounts of starting RNA such as that isolated from tissue biopsies and cultured cells. RNA amplification requires two purification steps; one following cDNA synthesis and a second following in vitro transcription. The most frequently used purification methodologies can be challenging to automate. RNAClean can streamline amplified aRNA production, because this single reagent can be used for purification of both the double stranded cDNA template and the in vitro transcribed aRNA. We performed aRNA amplification using the MessageAmp kit from Ambion. We compared purification using columns, RNAClean or a combination of the two (Figure 6). Products were separated by gel electrophoresis and quantified using a RiboGreen assay (Molecular Probes). Yields are expressed relative to that found when column purification was used for both purification steps. The highest yield of aRNA product was seen when RNAClean was used for both purification steps (Table 2).
Purifications based on SPRI chemistry are amenable to both manual and fully-automated formats. RNAClean has successfully been used in both tubes and 96-well plates. We used RNAClean to purify a 250 base RNA transcript in 96-well format. Figure 7 demonstrates the high level of consistency u sing RNAClean purification in a multi-well format. The simplicity of automating SPRI enables fully-automated purification of up to thirteen 96-well plates per hour on a Biomek FX.
RNAClean is a flexible platform that purifies high quality RNA/cDNA from a number of in vitro enzymatic reactions.
RNAClean efficiently and quantitatively recovers a wide range of RNA/cDNA fragment sizes.
Low volume elution allows for effective concentration for downstream application flexibility.
RNAClean can be performed manually or fully-automated, in a tube or plate-based format.
RNAClean simplifies aRNA amplification by using one easily-automated purification method for both cDNA and aRNA purification steps.
Barlow, J.J., Mathias, A.P., Williamson, R., and Gannack, D.B., (1963). Biochem. Biophys. Res. Commun. 13:61-66.
Hawkins, et al., (1994). Nucleic Acids Res. 22(21), 4543.
Maniatis, Sambrook, Fritsch, (1989). Molecular Cloning: A Laboratory Manual 2 ed. Vol. 3, Appendix E.12.
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