Multiplexed PCR-based Platform
Primera Biosystems (www. primerabio. com) developed a new technology for the simultaneous quantitative detection of multiple targets in a single sample. STAR, or Scalable Transcriptional Analysis Routine, integrates RT-PCR and capillary electrophoresis, allowing the detection of gene transcripts in a multiplex format. Amplicon size is used as an identifier for each target.
We found that microarrays were not very quantitative, and to find out which results were real and which werent, we had to use TaqMan. We thought there had to be a better way, where we can look at more genes that will be more quantitative and give us better data, says Elizabeth Garcia, Ph.D., director of assay development.
Specificity of PCR amplification is due to appropriate primer choice and reaction conditions. The hardest part of this assay is choosing the set of 20 primers that work well togetherthere are primer-primer interactions, explains Dr. Garcia. We are developing tools to make that much easier and redesigning the primers ourselves.
Since capillary electrophoresis allows accurate size determination of nucleic acids from 50 to 1,000 bases with the precision of one base, assays can be done simultaneously for many targets. Quantification capabilities are maintained equal to those seen with RT-PCR.
Invitrogen (www.invitrogen.com) developed several new products for gene expression analysis, ranging from sample preparation to RNA amplification and labeling. The primary product is the SuperScript III reverse transcriptase, says Patrick N. Gilles, Ph.D., R&D manager. This is more thermo-stable than the SuperScript II, resulting in longer, full-length cDNA synthesis with all of the splice variants. It also provides more reproducible array data with higher correlation coefficients.
The company has several SuperScript III labeling kits that incorporate Alexa Flors that are spectrally equivalent to cye dyes, but more stable and resistant to photobleaching and ozone degredation. One kit for direct labeling has direct incorporation into cDNA SuperScript Plus for DNA microarrays. The other is an indirect labeling kit that incorporates two nucleotides that can be conjugated to the dyes.
The unique part about this is that it allows you to use three-color experiments because we have three Alexa Flors with minimal overlap. This allows for a drastic reduction in the amount of required sample to obtain differential expression analysis. Since the Alexa Flors are more stable, it gives a better correlation coefficient that results in fewer false positives, states Dr. Gilles.
SuperScript III is also applied in the companys new RNA amplification kit. Starting with as little as 100 ng of RNA, it allows more full-length initial synthesis, so subsequent amplification is full-length and contains all the genes, even low-copy genes.
There are two new sample prep kits for obtaining small RNAs. The RiboMinus Transcriptome Isolation kit removes large ribosomal RNA but keeps the smaller RNA that can be used for labeling regulatory RNA. ChargeSwitch system eliminates many amplification inhibitors found in traditional purification (i.e., silica gel) methods. Binding is based on pH differencesat an acidic pH it will bind, at slightly alkaline pH it will dissociate. This allows for the binding of the total RNA preparation, whereas small RNAs would be lost through standard silica filters, explains Dr. Gilles.
The MAQC Project is working to provide quality control tools to avoid procedural failures and develop guidelines for microarray data analysis by providing large reference datasets and accessible reference RNA samples.