• Simplified Workflow
• All reactions occur in a single tube
• New Master Mix formulations reduce pipetting steps and hands-on time
• Shortened processing time due to optimized incubations and condensed procedure


• Improved Reaction Efficiency
• Start with as little as 20 ng total RNA and produce enough aRNA for GeneChip® analysis
• Start with 100 ng of total RNA (with a 4 hour IVT) and hybridize to a GeneChip in a single day


• Highly Consistent Results
• Less hands-on time means less opportunity to introduce errors
• Magnetic bead-based purification gives high yields with more consistent results

DNA microarrays are used to evaluate the expression levels of thousands of genes in a single experiment. High-density arrays are the method of choice for assessing gene expression on a genome-wide scale. One of the challenges associated with microarray technology, however, is that adequate amounts of labeled antisense RNA (known as cRNA or aRNA) are required. T7 linear amplification, commonly referred to as the Eberwine method, overcomes this challenge by simultaneously amplifying and labeling the RNA. This method is considered the gold standard for sample preparation in microarray-based expression profiling. The MessageAmp Premier and MessageAmp III RNA Amplification Kits are the next generation of Ambion biotin labeling/amplification kits to support microarray technology, such as the Affymetrix GeneChip® platform.

Workflow Improvements
Both the MessageAmp Premier and MessageAmp III Kits incorporate extensive improvements with a simplified workflow and more efficient reactions. The kits now consist of a set of master mixes, reducing the number of components, pipetting steps, and manipulations. Steps such as the Initial Primer Annealing and cDNA Purification have been eliminated (Figure 1). All steps of the biotin labeling/amplification process take place in a single reaction tube. This simplified workflow significantly shortens hands-on time, reducing operator-dependant errors and variability.

Improved Reaction Efficiency
The MessageAmp Premier Kit allows a researcher to perform expression profiling with as little as 20 ng of input RNA. The kit requires as little as 100 ng of total RNA producing enough labeled aRNA to hybridize to a GeneChip in a single day. Representative yields from the MessageAmp Premier Kit using HeLa total RNA are shown in Figure 2. Typical aRNA yield and median size (in nucleotides) for RNA from multiple tissue types are depicted in Figure 3.

MessageAmp Premier Kit or MessageAmp III Kit?
The MessageAmp Premier and MessageAmp III Kits differ only in their method of aRNA purification. The MessageAmp Premier Kit produces higher yields (approximately 25%) than the MessageAmp III Kit due to the efficiency of magnetic bead-based aRNA purification. The MessageAmp III Kit utilizes a traditional Glass Fiber Filter-based aRNA purification method.

Advantages of Magnetic Bead-based aRNA Purification
The MessageAmp Premier Kit employs a magnetic bead-based method for aRNA clean-up. Some advantages of this method are:

• Higher yields (up to 25%) than Glass Fiber Filter purification
• More consistent results
• No filters to clog
• Faster workflow
• Easier to handle using manual pipetting
• More amenable to high-throughput processing

Master Mix Formulations
Historically, Eberwine Linear Amplification kits have contained up to 24 different components. With the MessageAmp Premier and MessageAmp III Kits, these components have been condensed into a set of master mixes that significantly reduce hands-on time and pipetting steps. All master mix formulations are tested for quality using the highest standards in the industry.

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