NEW LabelStar Array System

The LabelStar Array System is designed for efficient labeling and cleanup of cDNA before array hybridization. Optimized reaction conditions result in high signal intensity, low background, and the identification of more true positives at low expression levels. The LabelStar System combines an easy-to-use system with high flexibility of labeling technique, choice of label, and amounts of RNA.

Features and benefits

• High signal intensity and low background — identification of true positives at low expression levels
• High specificity — labeling using 0.2–50 µg RNA
• Flexibility in choice of label — incorporation of any commonly used modified nucleotide
• Optimized labeling and cleanup procedure — reproducible, high-quality results
• Fast and easy method that provides reliable performance

Principle

The new LabelStar System offers a labeling and cleanup system that is adaptable to direct and indirect cDNA labeling using a wide range of modified nucleotides and amounts of RNA. The LabelStar System contains all reagents necessary for efficient and reproducible cDNA labeling and cleanup (excluding modified nucleotides). Using the LabelStar System, any commonly used modified nucleotide can be used in the labeling reaction, increasing flexibility.

Specifications
Amount of total RNA	0.2–50 µg
Amount of mRNA	0.2–5 µg
Modified nucleotides for cDNA labeling:
Cyanine-3/Cyanine-5-dCTP
Cyanine-3/Cyanine-5-dUTP
Biotin-dCTP or -dUTP
5-(3-aminoallyl)-2'-dUTP
32P-dCTP
33P-dCTP
Final cleanup reaction elution volume	10 µl

Procedure

The LabelStar System combines efficient cDNA labeling and cleanup (see flowchart "LabelStar Array Procedure"). The LabelStar System is comprised of two modules. The cDNA-labeling module contains LabelStar Reverse Transcriptase, dNTPs, RNase Inhibitor, and buffers and solutions for labeling. The cleanup module contains MinElute Spin Columns and buffers optimized for cleanup of labeled cDNA for microarray analysis.

During the LabelStar procedure, isolated RNA is treated with Denaturation Solution Plus to denature the RNA template and neutralize inhibitors copurified with RNA (see flowchart "LabelStar Array Procedure"). Modified nucleotides of choice (see Specifications) are incorporated during reverse transcription of the denatured RNA using LabelStar Reverse Transcriptase. For high signal intensity (see figure "High Signal Intensities Using the LabelStar Array Kit"), degradation of remaining RNA is necessary after the labeling reaction. The exoribonuclease activity of the LabelStar Reverse Transcriptase efficiently degrades RNA, eliminating the need for an extra RNA degradation step. Stop Solution LS stops the labeling reaction and reduces nonspecific binding of labeling components to the array, reducing background signal. The LabelStar System can be adapted to several labeling strategies (see figure "Different Labeling Strategies Using the LabelStar Array Kit").

Cleanup of labeled cDNA is performed with silicamembrane –based MinElute Spin Columns using a simple bind, wash, elute procedure. Optimized buffer sets and a novel spin column design ensure high recovery and high purity of labeled cDNA in very low elution volumes eliminating the need for further concentration steps. All contaminants from the labeling procedure, such as unincorporated labeled nucleotides or salts, are efficiently removed in the cleanup procedure.

High Signal Intensities Using the LabelStar Array Kit

cDNA labeling and purification was performed using 2 µg total RNA from mouse brain using the LabelStar Array Kit or a kit from Supplier A following the manufacturers instructions. The signal intensities of six spots were compared. Mean values of relative signal intensities were calculated (see bar chart). On average, signal intensities were 9-fold higher using the LabelStar Array Kit.

Applications

The LabelStar cDNA Labeling System is suitable for different labeling strategies in microarray analysis including:

• Direct cDNA labeling
• Indirect cDNA labeling
• Radioactive labeling
• Direct labeling after RNA amplification

Different Labeling Strategies Using the LabelStar Array Kit

The LabelStar System is adaptable to several labeling strategies. The labeling steps and cleanup performed using the LabelStar System are marked in blue.

LabelStar Array Kit

For efficient cDNA labeling and cleanup, prior to labeled cDNA array hybridization

操作手册: LabelStar Array Handbook 07/02 (PDF version, 177 KB)