( ...)Microarray Target Purification Kit and,,,the Microarray Target Synthesis Kit (T7)

As the human genome sequence project is completed, the vast amount of genomic information calls for methods that allow gene expression analysis on a genomic scale and speed up detection of significant sequence variations. The most suitable tools available for gene expression analysis are DNA hybridization arrays that allow researchers to examine differences in expression of hundreds or thousands of genes in parallel. That is why DNA arrays, on a variety of platforms such as macroarrays, microarrays or high-density oligonucleotide arrays make their way into different areas of functional genomics research. Arrays provide immobilized gene-specific sequences (probes) on different solid supports (e.g., nylon membranes, glass slides, or silicon/ceramic chips) and are hybridized with labeled targets that are copies of nucleic acids derived from various biological sources.



This requires reliable and efficient tools for preparation and labeling of targets: RNA needs to be extracted from small amounts of precious sample materials, and labeled either directly during cDNA preparation or via a linear amplification (100 to 1000 fold) with T7 polymerase producing labeled cRNA, or via polymerase chain reaction (PCR)-based amplification methods. This step must be highly reproducible and efficient to avoid differences in hybridization caused by biased amplification of target material.

Therefore, Roche Applied Science has developed a range of DNA microarray analysis products for expression profiling, suitable especially for very small amounts of starting material or when highest sensitivity is required. Each product is designed and tested in combination with the others to cover the critical workflow from RNA isolation to preparation of labeled targets. After each step, it is essential that residual impurities or nucleases are removed from the reaction products (single-stranded [ss] or double-stranded [ds] cDNA, cRNA or PCR products). Therefore, we have developed a nucleic acid purification kit that is universally applicable to the whole range of potential samples.

Product Description

cDNA Synthesis System

The well-established cDNA Synthesis System provides a convenient one-tube procedure for the synthesis of ds cDNA, starting even with small amounts of total RNA (120 g). Using the supplied Oligo ([dT24] T7 promoter) primer, cDNA is synthesized containing a T7 RNA polymerase promoter for the subsequent transcription reaction with T7 RNA polymerase.

Microarray Target Purification Kit

The Microarray Target Purification Kit is specifically designed for the purification of a variety of synthesized nucleic acids in the target labeling and amplification process, such as:

• ss cDNA after 1st strand cDNA synthesis
• ssDNA after cDNA labeling
• ds cDNA after 2nd strand cDNA synthesis
• PCR products
• cRNA after in-vitro transcription
  
  

The kit will remove residual nucleotides, primers, proteins, nucleases and other reaction components which will otherwise interfere in the workflow for highly sensitive microarray hybridization. The process does not require nucleic acid precipitation, phenol/chloroform extractions, or extensive handling of nucleic acids.

Microarray Target Synthesis Kit (T7)

The Microarray Target Synthesis Kit (T7) has been developed as a robust, reliable and highly reproducible tool for the preparation of large amounts of labeled cRNA when starting with only 105 106 cells for target preparation. Major features are:

• acceptance of low amounts of cDNA (obtained from 1 g total RNA)
• short reaction time of only 2 3 hours (Figure 2)
• high yields of cRNA in the range of 100 g
• compatible to a broad spectrum of hapten or fluorophore-labeled nucleotides
• cost-effective labeling due to optimized nucleotide concentrations and reaction conditions
  
  

Targets, synthesized and labeled with the Microarray Target Synthesis Kit (T7) have been tested on various commercially available DNA arrays. We have found that it is sufficient to use only one labeled nucleotide to get reliable results compared to double-labeling procedures in the majority of experiments.

Very often sample material is available in such small quantities (starting with less than 1,000 cells) that efficient target preparation requires PCR-based amplification methods. Within the Microarray Target Synthesis Kit (PCR), we have optimized a random primer-based PCR amplification procedure which allows the efficient and reproducible amplification of mRNA out of 50 ng total RNA.

The resulting PCR product can then be labeled by different methods, e.g., with the Microarray RNA Target Synthesis Kit (T7) to obtain single stranded cRNA.




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