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Mar 1, 2009 (Vol. 29, No. 5)

Quality Makes a Difference in RNA Prep

New Methodologies Are Being Counted On to Bring Order and Repeatability to Gene Expression

  • RNA Isolation

    “Quality, yield, and consistency are the three guiding principles of researchers in the field of RNA analysis,” explains Brian Kim, director for Ambion Technologies at Life Technologies. This means that, from the inception of a procedure, RNA profiles must be locked down. “The samples that investigators are targeting are heterogeneous, all different from one another,” he continues.

    Obtaining high-quality, intact RNA is the first and often the most critical step, and to this end Ambion produces a range of products. “To meet these needs, we produce kits for processing blood, paraffin-embedded tissue, cultured cells, and material from different organs,” he says.

    A long-standing product in the Ambion line is the RNAlater® Tissue Storage:RNA Stabilization Solution that allows the researcher to postpone RNA isolation indefinitely after tissue collection without sacrificing the integrity of the RNA. Dissected tissue or collected cells are dropped into the RNAlater solution that permeates the cells, stabilizing the RNA, and allowing biological materials to be stored for an indefinite period.

    The next stage of RNA processing, RNA isolation, is advanced by kits for total or poly(A) RNA isolation compatible with a range of plants, microorganisms, and animals. TRI Reagent® is a single, phenol-based, homogenous solution containing a combination of denaturants and RNase inhibitors, used in a single-step disruption and separation procedure. Another approach is the RNAqueous® Technology, a rapid, filter-based RNA isolation system that does not require the use of phenol, chloroform, or other toxic organic chemicals.

    Alternatively, total RNA may be isolated using Ambion’s ToTALLY RNA™ Kit, an alternative to the guanidinium thiocyanate/acid phenol:chloroform method but modified to reduce DNA, carbohydrate, heme, and other contaminants that can foul the preparations.

    The company’s products have features that make them especially useful for bacterial RNA purification, a task which presents its own idiosyncrasies. Speed is critical in the purification of prokaryotic RNA due to the short half-life of bacterial mRNA and the need to rapidly “freeze”  the mRNA expression profile. Easily lysed gram-negative bacteria may be pipetted directly into a boiling lysis buffer of choice (without even removing the culture medium), and RNA can be immediately extracted with the TRI Reagent.

    One of Ambion’s leading products, according to Kim, is the RecoverAll™ Total Nucleic Acid Isolation Kit for extraction of total RNA or DNA from formalin- or paraformalin-fixed, paraffin-embedded tissues. This kit enables the molecular analysis of archived tissue samples at both the genomic and gene-expression levels and is suitable for qRT-PCR, mutation screening, and microarray analyses. The four-hour protocol yields RT-PCR-competent RNA even for rare messages, Kim says.

    Eukaryotic mRNA is used for the construction of random-primed cDNA libraries. Removal of ribosomal and transfer RNA produces a 30-fold enrichment of a specific message. Ambion’s Poly(A)Purist™ Kits are designed for the isolation of the high-purity mRNA from total RNA, without sacrificing yield. The Poly(A)Purist and MicroPoly(A)Purist Kits include premeasured aliquots of oligo(dT) cellulose, whereas the Poly(A)Purist MAG Kit incorporates oligo(dT) magnetic bead-based purification.

    Isolation of mRNA from bacteria presents a special challenge, as they lack the relatively stable poly(A) tails found on eukaryotic messages. The MICROBExpress™ Bacterial mRNA Isolation Kit is designed to remove >95% of the 16S and 23S rRNA from total RNA of E. coli and other bacterial species.

    The kit is suitable for rapid mRNA purification from a broad spectrum of gram-positive and gram-negative bacteria.  In the first step of the MICROBExpress procedure, bacterial total RNA is mixed with an optimized set of capture oligonucleotides that bind to the bacterial 16S and 23S rRNAs. Next, the rRNA is removed from the solution using derivatized magnetic microbeads. The mRNA remains in the supernatant and is recovered by ethanol precipitation.

    Perhaps one of the most striking features of the RNA-isolation kits is their speed, in which whole segments of traditional isolation protocols are eliminated, and a molecule, ready for amplification, can be isolated in minutes. Examples include the Power SYBR® Green and Fast SYBR® Green Cells-to-CT™ Kits that employ a novel cell lysis and RNA-stabilization technology, according to Kim.

    This strategy completely eliminates the need for laborious and time-consuming RNA purification and integrates the lysis technology into a complete workflow including reverse transcription reagents and high-performance Power SYBR Green or Fast SYBR Green Master Mixes.

    Until recently, small RNAs were looked upon as a distracting contaminate, yet they are now recognized as critical features of the regulatory process. “Our mirVana™ miRNA isolation kit enables cancer researchers to isolate total RNA that contains small RNAs such as miRNA, siRNA, and snRNA that are often lost during traditional RNA isolation methods,” Kim states.

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