( ...)Critical factors for successful microarray and real-time PCR analyses

R&D Department
QIAGEN GmbH, Hilden, Germany Gene expression profiling at the RNA level has been significantly facilitated by microarray analysis and quantitative real-time RT-PCR. Microarray analysis offers the advantage of profiling expression levels of hundreds or thousands of genes simultaneously using asingle RNA preparation. Although real-time PCR analysis provides precise quantification over a wider dynamic range of expression levels, it is not suited for simultaneous analysis of large numbers of genes. Therefore, array analysis is often used as a tool to screen for target genes that are differentially expressed between biological samples, and quantitative real-time RT-PCR then provides precise quantification and validation of the microarray results. There are several prerequisites for successful and sensitive array analysis and reliable real-time RT-PCR. Critical steps include the preservation of the RNA expression profile in the biological sample as well as the efficiency of cDNA labeling and cleanup. Newly developed reagents and instrumentation enable increased sensitivity in microarray and real-time RT-PCR analysis. This article gives an overview of these critical factors and new developments. Sample collection and processing affects RNA expression pattern

A crucial step prior to analysis is stabilization of the RNA expression pattern in the biological sample. A biological samples RNA expression profile can dramatically change during collection and processing. This is due not only
to gene induction but also enzymatic degradation of RNA and/or down-regulation of genes (Figure 1). Immediate stabilization of the RNA and preservation of the RNA expression pattern is essential for accurate gene expression analysis.

Tissues stored without stabilization show significant changes in RNA levels. RNAlater RNA Stabilization Reagent stabilizes the RNA expression pattern in tissues even for prolonged times at ambient temperatures (Figure 2).

RNAlater TissueProtect Tubes Prevent RNA Changes
Changes in mRNA Levels
Using Traditional Methods Figure 2 Rat tissues (10 mg each) were excised and stored at room temperature for up to 4 hours in phosphate-buffered saline (PBS) or RNAlater RNA Stabilization Reagent (RNAlater). At the indicated times, RNA was isolated using the RNeasy Protect Mini Kit. Total RNA was used for quantitative RT-PCR using the QuantiTect Probe RT-PCR Kit and primers and probes specific for the transforming growth factor (TGF- ) gene or the tumor necrosis factor alpha(TNF- alpha) gene. Analyses were carried out in triplicate on the ABI PRISM 7700 Sequence Detection System. The change in threshold cycle (CT) is shown, relative to the CT at time zero. Figure 1 Schematic of changes in mRNA
levels during sample harvest and processing. Higher Signal Intensities with the LabelStar Array Kit RNAlater stabilized tissues can be conveniently and safely processed at room temperature without using dry ice or liquid nitrogen. New RNAlater TissueProtect Tubes provide RNAlater Reagent in easy-to-handle, reclosable tubes for immediate RNA stabilization at sample collection. This is especially important in fields such as molecular diagnostics, in order to immediately stabilize biopsies and other tissue samples directly after excision in a clinical environment, where liquid nitrogen is not allowed. Stabilized samples can be transported and easily processed at room temperature. QIAGEN provides a range of RNA stabilization reagents, including RNAprotect Bacteria Reagent for Grampositive and Gram-negative bacteria.

Integrated RNA stabilization and purification

The PAXgene Blood RNA System from PreAnalytiX provides a standardized system for integrated collection of blood samples with stabilization and purification of their RNA. RNeasy Protect Kits for animal tissues and RNeasy Protect Bacteria Kits integrate RNA stabilization and purification in a single kit. RNeasy silica-gelmembrane technology can also be fully automated with reaction setup on the BioRobot 8000, an ideal front-end tool for subsequent array or quantitative RT-PCR analyses.

Efficient cDNA labeling and cleanup for more true positive spots on microarrays

A common bottleneck to sensitive and reproducible array analysis is poor cDNA labeling efficiency and suboptimal cDNA cleanup. The new LabelStar Array Kit is specifically developed for microarray cDNA labeling, and provides a novel denaturation solution that resolves secondary structures in
the RNA template and neutralizes potential inhibitors associated with the RNA template. LabelStar Reverse Transcriptase can easily incorporate any labeled nucleotide during cDNA synthesis, giving high labeling efficiency (Figure 3). Selective binding to the cleanup column provides efficient removal of
non-incorporated nucleotides and of short, labeled extension products, which would otherwise contribute significantly to background in array analysis. The optimized cDNA labeling and cleanup procedure eliminates background and artificial signals, resulting in more true positive spots, even for genes with low expression.

Figure 3 cDNA labeling (with Cy5) and purification was performed using 2 g total RNA from mouse brain using the LabelStar Array Kit and a kit from supplier A, following the manufacturers instructions. The signal intensities of 6 spots were compared. Mean values of relative signal intensities were calculated. On average, signal intensities were ninefold higher using the LabelStar Array Kit.

Improved microarray sensitivity without amplification for analysis of true expression patterns

Successful gene expression profiling with microarrays is limited not only by RNA quality and cDNA labeling efficiency but also by the sensitivity of commercially available detection systems. Amplification of the target or signal is necessary when working with limited amounts of starting material, but this distorts the gene expression pattern due to the biases inherent in amplification methods. Two novel detection technologies overcome this limitation and enable analysis of small samples as little as 12 g total RNA and reflect the true picture of the expression pattern in the cell.

The HiLight Detection System (developed and manufactured by Genicon Sciences) improves the sensitivity of existing microarrays using Resonance Light Scattering (RLS) technology instead of fluorescence for detection. Suitable for a variety of commercial and self-spotted microarrays, the HiLight
Detection System enables microarray analyses with as little as 12 g total RNA. The stable, archivable signal allows multiple reads in order to detect low- and high-abundance mRNAs under optimal conditions and to normalize the signal (Figure 4).

The new SensiChip System (co-developed with Zeptosens) provides a complete microarray and detection solution with improved reproducibility and sensitivity in array experiments. Innovative planar wave guide technology (PWG) reduces background interference and dramatically increases the signal-to-noise ratio, providing exceptional sensitivity with as little as 1 g total RNA (Figure 5). The system includes customized SensiChip oligonucleotide arrays, which carry their own fluidics, and the SensiChip HybStation for standardized hybridization conditions. The SensiChip Reader enables high throughput analysis and includes software for image analysis.

HiLight Detection System Provides Increased Signal Intensity SensiChip System Reduces Background Figure 4 The HiLight Detection System uses biotin-labeled cDNA and HiLight RLS Particles SC coated with anti-biotin antibodies. The intense non-fluorescent signals enable simple and highly sensitive
detection of nucleic acids on microarrays. Figure 5 PWG technology provides excitation that is strictly confined to the
array surface. This results in high signal-to-noise ratios and improved sensitivity.

Validation of gene expression pattern with highly specific and sensitive quantitative real-time RT-PCR

The state-of-the-art technology for confirmation and quantitative analysis of expression levels is quantitative, real-time RT-PCR. QuantiTect Kits from QIAGEN provide high sensitivity and specificity in real-time PCR and RT-PCR. Optimized, ready-to-use master-mix solutions eliminate tedious optimization procedures, and the kits can be used on any real-time PCR instrument.

Quantitative real-time RT-PCR using the QuantiTect Probe RT-PCR Kit confirms the RNA abundance levels determined with the SensiChip System and the HiLight Detection System and provides highly accurate quantification. Signal-to-noise ratios in the array experiments correspond with expected
gene expression levels (Figure 6A and 6B), which also correlate with the precise quantification by real-time RT-PCR (Figure 6C, 6D, and 6E), demonstrating the reliability and reproducibility of the analyses.

Array Results Confirmed by Real-Time RT-PCR

Figure 6 RNA was isolated from murine brain tissue of inbred strain Black6 mice using the RNeasy Mini Kit, and 0.5 or 1 g total RNA was used for cDNA labeling and cleanup with the LabelStar Array Kit. Microarray analysis was performed with the SensiChip System using dCTP-Cy5 and dCTP-Alexa Fluor532 fluorescent dyes. Array analysis was carried out in parallel with the HiLight Detection System using dUTP-biotin with subsequent binding of HiLight RLS Particles SC. Signal-to-noise ratios (SNR) were determined for A the SensiChip System and B the HiLight Detection System. C Representative amplification plot for quantification of TCF4 using one-step quantitative real-time RT-PCR with the QuantiTect Probe RT-PCR Kit. D Representative standard curve using the QuantiTect Probe RT-PCR Kit and 102.106 copies of an in vitro transcript of TCF4. E Copy numbers for all three genes.

Functional analysis of differentially regulated genes using novel siRNA technology

In addition to gene expression analysis using arrays or real-time RT-PCR, QIAGEN offers an advanced gene silencing method for functional gene analysis. This method makes use of small interfering RNA molecules (siRNA), synthesized by QIAGEN. siRNA triggers sequence-specific mRNA degradation leading to posttranscriptional silencing of a target gene. These double-stranded siRNA molecules can be efficiently transfected into eukaryotic cells using TransMessenger Transfection Reagent.

Conclusions

• QIAGEN provides all you need for highly sensitive array analysis - from sample stabilization and preparation to customized chips and two highly sensitive array detection technologies.
  
• For state-of-the art quantification and confirmation of array results, QIAGEN has everything you need - from RNA stabilization and purification to primers, probes, enzymes, and QuantiTect Kits for quantitative PCR and RT-PCR.

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