Synthesize High Yields of Biotinylated aRNA
Ambion now offers an improved MessageAmp™ II Kit with biotin-11-UTP. MessageAmp II-Biotin Enhanced provides a complete single round amplification kit for biotin array analysis. The biotin modified UTP and ratio of labeled to unlabeled UTP were selected based on superior performance and aRNA yield. The biotinylated nucleotide is supplied as a pre-mixed NTP at an optimized ratio, providing convenient,
T7 RNA Polymerase Linear Amplification is the Gold Standard
Higher Yields of Labeled aRNA; Only 1 Round of Amplification
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 µg) 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, 50–100 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 MessageAmp™ II-Biotin Enhanced Kit (MA II-Biotin) or another manufacturer’s 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 manufacturer’s 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 above 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).
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
Robert Setterquist, Mike Wilson, Charles Johnson, Shika Agarwal, Sharmili Moturi • Ambion, Inc.