MessageAmp™ II: Use Less RNA for Array Analysis
Better Reverse Transcription and cDNA Synthesis
In developing MessageAmp II, the reverse transcription and second strand cDNA synthesis steps, both critical for generating high yields of full-length labeled aRNA, were optimized. Major improvements include the development of ArrayScript™ M-MLV RT. ArrayScript is an engineered M-MLV reverse transcriptase that synthesizes exceptionally high yields of full-length cDNA unattainable with wild type enzymes. The second strand cDNA synthesis reaction was also further optimized to convert the cDNA to double-stranded cDNA with high efficiency. As a result, as little as 100 ng of total RNA in a single round of amplification with MessageAmp II generates enough aRNA for array analysis using Affymetrix GeneChip® Arrays. Other commercially available products for RNA amplification require two rounds of amplification at this level of RNA input.
Together, these improvements result in more efficient conversion of mRNA into longer double-stranded cDNA templates. This, in turn, enables shorter in vitro transcription (IVT) reaction times and reliable expression analysis from small RNA samples (less than 1 µg of total RNA). The MessageAmp II improvements also have an impact when two rounds of amplification are employed, allowing more robust amplification of total RNA amounts from 10 ng to as little as 100 pg.
Here we provide aRNA yield data and guidelines for the number of amplification rounds needed based on the amount of input total RNA and the RNA source used in the reaction.
Factors Affecting cRNA Yield
Figure 1. MessageAmp™ II aRNA Amplification Yields. Yields of aRNA (µg) amplified using the MessageAmp II aRNA Amplification Kit from six different RNA sources using total RNA inputs of 50–3000 ng. Yields shown are the average of duplicate reactions. The IVT incubation time was 4 hours for all samples. Purified aRNA concentrations were measured using a NanoDrop® spectrophotometer.
*Universal Human Reference RNA (Stratagene)
The IVT reaction time also has an impact on aRNA yield from MessageAmp II reactions. The yield of aRNA increases with increasing RNA input and IVT time. As a rule, most amplification reactions containing between 100–1000 ng input total RNA, incubated overnight (14 hr IVT), will produce sufficient aRNA for any microarray platform (at least 10 µg). While a minimum of 4 hours incubation time is recommended, yields obtained from 2–3 hour incubations are often sufficient, and >500 ng input RNA in a 2–4 hr incubation will typically produce enough aRNA for microarray analysis.
One or Two Rounds of Amplification?
It should be noted that the size range of aRNA produced by amplification also varies with input amount. A typical trace of the aRNA produced from 1 µg and 100 ng of input RNA after 1 round of amplification is shown in Figure 2. Figure 3 shows bioanalyzer traces of aRNA produced after two rounds of amplification for several different input RNA amounts, and the bar graph in Figure 4 shows the aRNA yields for those inputs. Note that while the size of the resulting aRNA is very consistent for a given amount of total RNA input, at the very low input amounts used for two rounds of amplification, yields will vary from user to user and from RNA sample to sample.
It is often the case that total RNA yield from a set of tissue or cell samples varies considerably. Some samples may yield 1 µg of total RNA such that only one round of amplification is needed, whereas others may only yield 50 ng, thus requiring two rounds of amplification. For consistency within and between experiments, all samples in the study should be amplified with either one or two rounds of amplification; i.e. the amplification parameters for the smallest amount of input RNA in the sample set should be chosen for the entire study. Figure 4 shows some empirical data that may be useful in deciding whether one or two rounds of amplification are needed for samples smaller than 100 ng. In general, one round of amplification will not yield sufficient amounts of aRNA if RNA input levels are below ~50–75 ng.
Figure 4. aRNA Yields After Two Rounds of Amplification. Plot of typical aRNA yields (triplicates determined by UV absorbance) for 1000, 100, 50, and 10 pg of input HeLa cell total RNA after two rounds of amplification. In vitro transcription reactions were performed for 16 hr for both rounds.
An Improved Kit for aRNA Amplification
The MessageAmp II aRNA Amplification Kit contains all necessary reagents and components 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.