RNA Purification

We typically use RNA purification by the Trizol method (Invitrogen, following the manufacturer s instructions), which has the advantage over column-based methods that it can purify small amounts of RNA and retain miRs. Purified RNA is dissolved in RJNAse-free water and stored at —80°. RNA quality is assessed on an Agilent Bioanalyzer (Agilent Technologies) or by gel electrophoresis. 

The LiClprecipitation is necessary to remove any residual heparin, which may intefere with the labeling reaction. Some vendors (e.g., Ambion and Qiagen) sell RNA purification columns that should remove heparin. We have not tested any of these yet. 

The RNA purification step is necessary for removal of DNA, unincorporated nucleotides, and transcription byproducts. DNA must be removed because it can obstruct subsequent reverse transcription and PCR (Section 7.3.1.6). This can be achieved by DNase I digestion and/or by denaturing polyacrylamide gel electrophoresis (PAGE, 6%-10 % polyacrylamide, 7 M urea) [14]. Unincorporated nucleotides and transcription byproducts are best removed by denaturing PAGE. 

Always make sure to thoroughly clean the columns after each use, and if columns are not to be used in the next few days, store them in 20-30% ethanol at 4 °C. It is best to keep several Ni-NTA columns that are dedicated to RNA purification. Using these columns for protein purification can introduce many foreign proteins to the columns, potentially including RNases. 

Two other powerful methods for native RNA purification, one based on size-exclusion chromatography and the other using affinity purification, have been published in the past. The former employs separation of the different species in a transcription reaction (NTPs, small abortive transcripts, the transcribed RNA, and the plasmid DNA) based on size rather than charge (Kim et al., 2007 Lukavsky and Puglisi, 2004 McKenna et al., 2007). 

As in TRI reagents, such as TRIZOL reagents used for RNA purification from cells and tissues... 

ConnoUy MA, Clausen PA, Lazar JG. RNA purification. In Dveksler GS, ed. PCR Primer A laboratory manual. 2nd ed. New York Cold Spring Harbor Laboratory Press, 2003 117-33. 

RNA Purification Total RNA was isolated from T. spiralis mnscle larvae, adult forms and newborn larvae, and C. elegans adnlt worms, and LI, L3 and daner larvae, using TRIZOL ultra pure reagent from GibcoBRL, according to manufacturer s protocol. 

RNase-Free DNase Set, on-column digestion of DNA to be used during RNA purification (Qiagen). Set includes DNase I (1500 Kunitz Units), DNase buffer RDD, and nuclease-free water. 

PCR-compatible aqueous chemistiy in the extraction method. The utility of the device for purification of RNA was also verified by extraction of RNA from a dilute semen sample, with the resulting RNA amplified using reverse transcription (RT)-PC3l. The vrSPE-SPE device reliably yielded a volume reduction for DNA and RNA purifications by the order of 50- and 14-fold, respectively and both were compatible with downstream PC31 analysis. In addition, purification of all samples cmisumed less reagents (by 2.6-fold) than traditional purification methods (Pig. 3). 

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