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
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.
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.
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 qu antification 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 lea ding to posttranscriptional silencing of a target gene. These double-stranded siRNA molecules can be efficiently transfected into eukaryotic cells using TransMessenger Transfection Reagent.