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RNA & cDNA Purification Using RNAClean

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).


Consistent Recovery
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
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