MessageAmp™ II: Use Less RNA for Array Analysis
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Better Reverse Transcription and cDNA Synthesis
Ambion’s MessageAmp™ aRNA Amplification Kit was the first commercially available Eberwine-based RNA amplification kit. The next generation MessageAmp II aRNA Amplification Kit (patent pending) incorporates extensive improvements to the original kit.
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.
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
Factors such as tissue source and disease state, mRNA content (0.1–3%), and starting amount and quality of total RNA will all affect the yield of aRNA from any amplification-labeling procedure. Figure 1 shows typical yields of aRNA amplified from total RNA isolated from six different tissues using the MessageAmp II aRNA Amplification Kit. RNA input amounts ranging from 50–3000 ng were tested from each source. These data show some general trends found to be reproducible. For example, while aRNA yield generally increases with increasing amounts of input RNA, adding more than 1 µg of HeLa S3 RNA does not significantly increase aRNA yield. Also, kidney RNA, which generally produces less aRNA than other tissues, does not produce proportional increases in aRNA yield with increasing amounts of input RNA up to above 1 µg. Figure 1 also provides a useful benchmark for understanding the relationship between the amount of aRNA produced and the source and quantity of input RNA.
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.
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?
Based on experimental requirements and the amount of sample available, researchers must determine whether one or two rounds of amplification are appropriate. Microarray studies involving small samples or low mass amounts of sample RNA demand a high level of amplification (106-fold or greater). In situations like this, it is often beneficial to use the lowest level of total RNA input that generates enough cRNA for array hybridization. As little as 100 pg of total RNA can be used in the MessageAmp II procedure, and with two successive rounds of amplification, enough aRNA can often be produced for microarray analysis. Of course the amount of input RNA needed will depend on the amount of aRNA required for a particular microarray platform, and whether aRNA is also needed for replicates, follow up validation, or for archiving samples. We recommend considering two rounds of amplification when less than 100 ng of input total RNA is used.
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.
Figure 2. Size of cRNA Produced from Different RNA Inputs. Bioanalyzer electropherogram of amplified RNA (single round amplification) derived from 1.0 and 0.1 µg input HeLa cell total RNA. The mRNA Smear Assay was run in order to calculate the average size distribution. The center red line is the midpoint of the curve at 1030 nt for the 1.0 µg input trace. The 0.1 µg input midpoint size was 980 nt.
Figure 3. cRNA Derived From Different Total RNA Inputs. Bioanalyzer electropherogram of amplified RNA derived from 1000, 100, 50, and 10 pg input HeLa cell total RNA (two rounds amplification). The mRNA Smear Assay was run in order to calculate the average size distribution. The center red line is the midpoint of the curve at 630 nt for the 10 pg input trace and varies from 450 to 630 nt for the input range shown.
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.
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 inclusion of ArrayScript in conjunction with the optimization of the second-strand cDNA synthesis reaction in the MessageAmp II Kit results in increased conversion of mRNA into full-length, double-stranded cDNA templates. The kit also uses Ambion’s MEGAscript® IVT technology, generating enough cRNA for GeneChip analysis from as little as 100 ng of input RNA in a single round of 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.
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.
