Highest amplification in a single roundup to 10,000-fold for most samples
Enough aRNA for Affymetrix GeneChip analysis from just 50 ng total RNA
Optimized NTP mix containing biotinylated UTP reduces set-up time and maximizes labeling
Reduce costs by 45%
T7 RNA Polymerase Linear Amplification is the Gold Standard
Microarrays measure the expression levels of
thousands of genes. One limitation of this technology,
however, is the need for large amounts of labeled target RNA
for hybridization to the microarray. This limitation is
overcome by using a T7 linear amplification protocol commonly
referred to as the Eberwine Method. This method most
accurately maintains the initial mRNA expression profile and
has been the gold standard for microarray target preparation
. Biotin, incorporated as biotinylated nucleotide
phosphates during the in vitro transcription (IVT) step, is
the preferred label for most commercial microarray platforms.
The highly specific and efficient binding of streptavidin-dye
Cy dyes) to biotinylated RNA/oligonucleotide probe hybrids is the basis of highly sensitive fluorescence detection. Ambion has incorporated biotin labeling reagents into the MessageAmp II Kit to provide a complete microarray aRNA preparation kit, the MessageAmp II-Biotin Enhanced Kit.
Higher Yields of Labeled aRNA; Only 1 Round of Amplification
Both biotin incorporation and further optimization of the IVT reaction has resulted in increased sensitivity of microarray transcript detection. The increased sensitivity allows the use of less input RNA-50 ng of input RNA in a single round of amplification provides enough aRNA for array analysis. Amplified RNA yields derived from nine different tissue and cell sources and four different input amounts are shown in Figure 1. While length of the aRNA synthesized will largely depend on RNA sample quality, the cDNA and IVT reaction conditions in this kit produce longer biotinylated aRNA compared to the market leader. Figure 2 shows comparable Agilent bioanalyzer scan results from labeled aRNA produced with MessageAmp II-Biotin Enhanced Kit and unlabeled aRNA produced with MessageAmp II Kit. The resulting improvements also give the MessageAmp II-Biotin Enhanced Kit a higher number of Present calls than the market leader (Figure 3).
Figure 1. aRNA Yield from Nine Different Sources. Four different amounts of total RNA from nine different sample types were amplified using the MessageAmp II-Biotin Enhanced Kit. Average aRNA yields from triplicate reactions are shown for 4 or 14 hr in vitro transcription (IVT) reactions. These data are useful for determining both the amount of total RNA needed to obtain enough labeled aRNA for array hybridization (typically ~10 g) and the optimal length of IVT incubation. Note that there is a ~3-fold difference in aRNA yield between some samples. With most RNA sources, 50100 ng of input total RNA amplified with the MessageAmp II-Biotin Enhanced Kit using a 14 hr IVT incubation will yield enough labeled aRNA for microarray hybridization.
Figure 2. Electropherogram of Biotin-labeled vs. Unlabeled aRNA. Identical HeLa cell total RNA samples (1000 ng) were amplified with either the MessageAmp II-Biotin Enhanced Kit to produce biotin-labeled aRNA or the MessageAmp II Kit to produce unlabeled aRNA. The IVT reactions were carried out for 4 hr. Equivalent mass amounts from each reaction were analyzed on an Agilent bioanalyzer. The shift in aRNA size from the MessageAmp II-Biotin Enhanced sample results from the incorporation of biotin into the aRNA.
Figure 3. Number of Present Calls Detected on Affymetrix Human Genome Focus Arrays. The indicated amounts of total RNA from HeLa cells was amplified in triplicate using either the Mess ageAmp II-Biotin Enhanced Kit (MA II-Biotin) or another manufacturers aRNA labeling kit (Kit A). Two IVT incubation times (4 and 14 hrs) were tested with 1000 ng of total RNA input. Amplification of as little as 50 ng total RNA with the MessageAmp II-Biotin Enhanced Kit produced a comparable number of Present calls as 1000 ng input RNA that was amplified with Kit A. Furthermore, the amplification reaction can be completed in one day.
Hybridization of the biotinylated aRNA produced from the MessageAmp II-Biotin Enhanced Kit to Affymetrix GeneChip Arrays was used to assess the sensitivity and reproducibility of the method and reagents. Increased sensitivity can be measured several ways. One indication of sensitivity is the number of Present calls calculated by the GeneChip Operating Software (Figure 3). Additional analysis of the distribution of signal intensities indicates that the new MessageAmp II-Biotin Enhanced Kit produces a higher mean value. This means that a there was a higher signal intensity across the array (Figure 4). This results in more reproducible array results and increased sensitivity of lower expressed transcripts.
Figure 4. Higher Signal Intensity Distribution on Affymetrix Human Genome Focus Arrays. Distributions of log2 signal intensity values were plotted for the MessageAmp II-Biotin Enhanced Kit and another manufacturers amplification kit (Kit A). The median values for each of the MessageAmp II-Biotin Enhanced plots were similar for total RNA inputs ranging from 50 to 1000 ng. In contrast, signal intensities produced by Kit A at the 1000 ng total RNA input level was markedly reduced. The box and whiskers plot ab ove each histogram indicate the median value (vertical center line), interquartile range (left and right sides of box), and the 10% and 90% quantiles (ends of line). Each plotted distribution represents the average log2 signal intensity of triplicate target preparations and hybridizations. IVT reactions were carried out for 4 or 14 hrs as indicated. The data were normalized within each group using Robust Multichip Average (RMA) (See sidebar, Data Analysis: Robust Multichip Average (RMA) and Affymetrix Algorithms).
Biotin-11-UTP serves as the label in the MessageAmp II-Biotin Enhanced Kit. Only a single biotinylated NTP is used because studies at Ambion and elsewhere  showed that in previous dual labeling experiments using biotin-UTP and biotin-CTP, the signal contributed by biotin-CTP was minimal, and relative signal intensity between genes was not altered with the use of a single biotin UTP label.
While various linker arm sizes (e.g., Biotin-11-UTP, Biotin-16-UTP) did not affect modified UTP incorporation into aRNA during IVT, the longer linker arms slightly impeded subsequent purification of the aRNA. Biotin-11-UTP provided the highest yields of aRNA.
The ratio of biotin UTP to unlabeled UTP was optimized to provide the highest yield of aRNA and the highest signal:noise on Affymetrix GeneChip Arrays. A fairly wide range of concentrations of Biotin-UTP (25 60%) can be used for acceptable detection, whereas at lower concentrations the signal drops dramatically, and at higher concentrations both the noise increases and the aRNA yield decreases.
Ambion realizes that microarray users may prefer to continue using their own biotin-labeling method, or to experiment with different labeling conditions. To accommodate this we offer two Biotin-UTP nucleotides (Biotin-11-UTP and Biotin-16-UTP) separately, which can be used with the standard MessageAmp II Kit.
MessageAmp II-Biotin Enhanced Kit Format
The MessageAmp II-Biotin Enhanced Kit provides enough reagents for 20 single round amplification reactions. It can be used in combination with the MessageAmp II Kit for small samples that require 2 rounds of amplification (MessageAmp II for the first round and MessageAmp II-Biotin Enhanced in the second round for labeling). Biotin-UTP is provided premixed with the other rNTPS for convenience and consistent, optimized labeling.
Robert Setterquist, Mike Wilson, Charles Johnson, Shika Agarwal, Sharmili Moturi Ambion, Inc.
Cat# Product Name Size 1334 MEGAscript T7 Kit 40 rxns 1751 MessageAmp II aRNA Amplification Kit 20 rxns 1791 MessageAmp II-Biotin Enhanced Single Round aRNA Amplification Kit 20 rxns 8450 10 mM Bio-11-UTP 25 l (250 nmol) 8451 75 mM Bio-11-UTP 30 l (2250 nmol) 8452 10 mM Bio-16-UTP 25 l (250 nmol) 8453 75 mM Bio-16-UTP 30 l (2250 nmol)