RNA Amplification Just Got Better: MessageAmp™ II
Gene arrays simultaneously measure the expression levels of thousands of genes in a single experiment and have become the most widely used method for assessing gene expression on a genome wide scale. The primary limitation of this technology is the requirement for large amounts of RNA for array hybridization. This is especially a problem when working with limited samples, such as small biopsies, mRNA-deficient cells and tissues, primary cell cultures, and laser capture microdissection (LCM) samples. This hurdle is usually overcome by using T7 RNA polymerase driven transcription of cDNA obtained from total RNA samples to generate sufficient amounts of amplified antisense or copy RNA (aRNA or cRNA). This amplification method is commonly referred to as the "Eberwine method" , "IVT method" or the "aRNA or cRNA method".
Ambion's new MessageAmp II aRNA Amplification Kit (patent pending) incorporates extensive improvements to the original MessageAmp aRNA Amplification Kit. Improving amplification involves more than simply increasing the final yield of labeled cRNA. Consistency with other amplification methods is also important so data can be compared across methods. Microarray expression profiling data from MessageAmp II and the Affymetrix recommended protocol for GeneChip™ analysis show a high level of concordance. The new MessageAmp II Kit provides a wide dynamic range of RNA input (100 pg-5 µg), and a single round of amplification on samples as small as 100 ng generates sufficient cRNA for GeneChip analysis.
Improved Yields of Full-length cDNA
Higher Quality cDNA. Ambion's new ArrayScript produces higher yields of full-length cDNA compared to other reverse transcriptases (Figure 1). Figure 2 demonstrates that up to twice as much cRNA is synthesized when first-strand cDNA is generated with ArrayScript compared to wild type M-MLV, AMV, or other engineered M-MLV reverse transcriptases when low amounts of total RNA are used (~100 ng). This improved performance may eliminate the need for two rounds of cRNA amplification when working with small samples, thus reducing reagent cost and reaction time.
Second-strand cDNA Synthesis. The second-strand cDNA synthesis reaction has also been optimized specifically for the first-strand cDNA products generated with ArrayScript. This results in maximal conversion of first-strand cDNA into full-length double-stranded cDNA templates, thus increasing the average length of cRNA synthesized using MessageAmp II.
Figure 1. ArrayScript™ Reverse Transcriptase Synthesizes Intact cDNA Products as Long as 9 kb. cDNA synthesis was performed with 100 U of either ArrayScript RT, SuperScript™ II (SSII), SuperScript™ III (SSIII), or wild type (wt) M-MLV RT in a 20 µl reaction volume containing a synthetic, poly(A)-tailed 9 kb transcript (1 µg), 1X RT buffer, 0.5 µM oligo(dT) primer, 0.5 mM dNTPs, 40 U RNase Inhibitor, and 0.1 µl [α-32P]dATP. Reactions were incubated for 1 hr at 42ºC and quenched with EDTA. The cDNA products were resolved on a 1% denaturing agarose gel and detected with a phosphorimager.
Figure 2. ArrayScript™ Reverse Transcriptase Produces up to Twice the aRNA Yield in MessageAmp™ II. A comparison of ArrayScript RT (AS; 200 U), SuperScript II (SSII; 200 U); wild type M-MLV RT (200 U), and wild type AMV RT (10 U) in MessageAmp II conditions demonstrates that ArrayScript outperforms other commercial RTs. 90 ng HeLa-S3 total RNA (28S/18S ratio = 1.7) was heat denatured in the presence of T7 Oligo(dT) at 70°C for 10 minutes. Reverse transcription was initiated with the RT enzyme and incubated at 42ºC for 2 hr in an air incubator. Following first strand cDNA synthesis, DNA polymerase was added for second strand synthesis, which was incubated at 16ºC for 2 hr in a thermocycler (heated lid option turned off). The double-stranded cDNA was then filter purified and added to a 4 hr in vitro transcription reaction. Last, the resulting aRNA was purified by glass filter and eluted in nuclease-free water for analysis by Agilent 2100 bioanalyzer and NanoDrop® Spectrophotometer.
An Improved Amplification Kit
The MessageAmp II aRNA Amplification Kit contains all the necessary reagents for first-strand cDNA synthesis, RNase H digestion, second-strand synthesis, cDNA purification, in vitro transcription and aRNA purification. Reagents for 20 reactions and a detailed Instruction Manual are included.
Figure 3. Replicate Analysis of Two Amplification Protocols. Normalized array results are plotted from replicate amplifications. Both MessageAmp™ II (top) and Affymetrix® standard protocol (bottom) were used to amplify with the identical total RNA samples (1 µg), and equivalent amounts of cRNA were hybridized on matched lot Human Genome Focus arrays. All IVT reactions were performed for 4 hours. Correlation values are included and indicate a measure of reproducibility with both methods.