Achieving Seamless RNA Analysis

April 15, 2006 (Vol. 26, No. 8)

Single-Source Systems, Sample Prep, RNA Amplification, and RNAi Seen as Key Trends

Good-quality RNA impacts downstream analysis and results, explains Lucy Sha, marketing director for gene and protein discovery at GE Healthcare (www.gehealthcare.com). &#8220Sample preparation is critical, and the entire process of RNA analysis should be seamless, without bottlenecks,&#8220 Sha adds.

William Dietrich, product manager for RNA analysis at Stratagene (www.stratagene.com), agrees, adding, &#8220Trends in RNA analysis include getting value from a product&#8212saving time and money and simplifying the workload and process&#8212by getting complete solutions from one supplier.&#8220

At GE Healthcare, &#8220we&#180ve taken the products we&#180ve created for nucleic acid amplification and purification and brought the same expertise to sample preparation,&#8220 Sha says. &#8220The objective is to let researchers focus more time and energy on the creative aspects of their work.&#8220

Total RNA isolated using the GE Healthcare RNAspin Isolation kits is consistently of high quality and good yield, Sha notes. The quality is sufficient for use in transcription profiling workflows like microarray analysis, which takes the total RNA through cDNA synthesis and labeled-cRNA amplification to loading on microarray and expression analysis.

cRNA generated using this total RNA is also reproducibly of high quality, according to the company. Individual total RNA samples are highly uniform in their quality and transcript population representation, which is crucial for microarray experiments.

Sample preparation for RNA isolation and gene-expression measurement is a key area for Stratagene. One of its products enables gene-expression researchers to lyse and stabilize cells and go directly to the RT-PCR process.

Stratagene&#180s SideStep cDNA synthesis and QRT-PCR products that use the company&#180s Brilliant QRT-PCR technology are designed to deliver efficient cell lysis, RNA stabilization, and sensitive quantitative gene expression analysis without RNA purification. Enabling researchers to screen multiple samples first, they need only purify the most relevant, interesting RNA samples, saving over 70&#37 of the cost and 60&#37 of the time it takes to column purify RNA, according to Dietrich.

SideStep lysis and stabilization technology stabilizes RNA for up to six months using a single-tube format, minimizing RNA loss and degradation. The easy-to-use, nontoxic method accommodates a wide range of cell input, making it a flexible method for immediate gene-expression measurement from cells.

Applications include detecting gene expression of siRNA-induced knockdown experiments, gene-expression measurement in real time using SYBR or probe-based chemistry, miRNA detection, low-abundance message detection, mRNA enrichment directly from cells, producing high-quality, full-length QPCR-grade cDNA directly from cells, and analyzing laser microdissected cells from fresh or frozen tissues.

RNA Electrophoresis and Analysis

Cambrex Bio Science Rockland (www.cambrex.com) offers a complete line of ready-to-use products for RNA electrophoresis and analysis, according to Mary Riley, product manager. Gels, buffers, stains, and markers are manufactured to strict quality-control standards and guaranteed RNase-free.

Cambrex&#180 Reliant Precast RNA Gels and Latitude RNA Midigels are ready-to-use RNase free agarose gels suitable for a variety of RNA applications, including Northern blotting and analysis of RNA transcripts. Both come in a kit that contains all of the components for running an RNA gel in one package&#8212precast RNA gels, sample buffer, RNA marker, AccuGENE 10X MOPS buffer, and GelStar Nucleic Acid Stain.

RNA markers consist of RNA transcripts from 0.5 to 9 kb. These RNA standards may be denatured with standard procedures and can be visualized on Northern blots with labeled lambda sequence. GelStar Nucleic Acid Stain is a highly sensitive fluorescent stain for detecting both DNA and RNA that can be added to an agarose solution prior to casting or to post stain gels.

&#8220This complete system provides a clean, closed system for the ultimate in assurance and performance,&#8220 Riley says.

&#8220RNA amplification is critical to microarray or real-time PCR of low expressed messages or low starting templates,&#8220 explains Guy Afseth, director of marketing at Epicentre Biotechnologies(www.epiboio.com). &#8220Researchers might want to do RNA amplification from small sizes&#8212from flow cytometry, laser dissections, or small samples with a lot of background.&#8220

Amplification Kits

Epicentre&#180s TargetAmp amplification kits produce microgram amounts of various kinds of RNA. Available in various configurations, they are optimized for use with reverse transcriptase.

The company&#180s MessageBooster cDNA Synthesis Kit for qPCR is designed to greatly improve qRT-PCR sensitivity, accuracy, and reproducibility of even low-abundance transcripts from as little as one cell. A MessageBooster Kit reaction amplifies the Poly(A)RNA in a total RNA preparation of 1 to 50 cells and then converts the amplified RNA to single-stranded sense cDNA that is ready for qPCR, Poly(A) RNA amplification and subsequent cDNA synthesis.

The number of qPCRs that can be obtained using cDNA produced by a MessageBooster reaction depends on the amount of total RNA added to the MessageBooster reaction and the abundance of the transcript(s) of interest.

Bio-Rad&#180s (www.biorad.com) Aurum total RNA kits produce total RNA for major applications. After initial sample collection and disruption, cells are homogenized in either the Aurum total RNA lysis buffer or PureZOL RNA isolation reagent. The addition of alcohol, three wash steps, and an elution step follow. Purified RNA is recovered in high yield by saturating the membrane with a warmed elution solution. Vacuum-compatible protocols and a choice of preparative formats streamline sample preparation of total RNA. Aurum total RNA kits include quick protocol using silica membrane binding, RNase-free reagents and plasticware, and high yields of RNA from many sample types.

RNAi

Another hot topic in RNA is RNAi, which Peter Welch, director of R&D at Invitrogen (www.invitrogen.com), describes as &#8220a way of knocking down gene expression, first described in lower organisms and accomplished in mammalian organisms several years ago. Short dsRNAs enter the silencing complex in the cell, cleave, and destabilize, with no protein expression.&#8220

According to Christine Rondione, director of research of metabolic diseases at Roche (www.roche.com), &#8220RNAi is being used for target identification and validation and cell-based assays to determine the phenotype of cells. In metabolic diseases, you can&#180t simply measure apoptosis. We&#180re looking into RNAi to develop target identification for metabolic diseases, diabetes, obesity, oncology, and hepatitis, to eventually develop therapeutics.&#8220

Invitrogen Stealth Select RNAi sequences are ready-to-order duplexes to thousands of human genes. Stealth Select RNAi uses the same Stealth RNAi modified chemistry that results in complete and effective knockdown using RNA interference, minimizing concerns about unwanted off-target and nonspecific effects.

The Stealth Select 3 RNAi Set provides three nonoverlapping sequences to each gene, supplied individually, so that experiments can be validated using independent sequences and to comply with publication requirements. It also reduces off-target effects because of the application of an advanced specificity search in addition to BLAST and provides complete knockdown success.

Designed for researchers new to RNAi, Ambion&#180s (www.ambion.com) Silencer siRNA Starter Kit provides reagents, protocols, and instructions for siRNA transfection and gene-knockdown experiments. Included is a lipid-based transfection agent, siPORT NeoFX, that delivers siRNAs to a broad range of cell lines. siPORT NeoFX can be used for reverse transfection, a streamlined protocol, as well as with standard transfection protocols. This reagent is not sensitive to serum and efficiently transfects low concentrations of siRNA.