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 l reaction volume containing a synthetic, poly(A)-tailed 9 kb transcript (1 g), 1X RT buffer, 0.5 M oligo(dT) primer, 0.5 mM dNTPs, 40 U RNase Inhibitor, and 0.1 l [a-32P]dATP. Reactions were incubated for 1 hr at 42C and quenched with EDTA. The cDNA products were resolved on a 1% denaturing agarose gel and detected with a phosphorimager.
Double the Yield in RNA Amplification
One of the most rigorous applications for an RT enzyme is RNA amplification from low amounts of total RNA. In this application, first-strand cDNA generated with ArrayScript resulted in up to twice the yield of amplified RNA compared to cDNA synthesized using wild type M-MLV, AMV, or other engineered M-MLV reverse transcriptases (Figure 2). When starting with low amounts of total RNA, ArrayScript often eliminates the need for two rounds of amplification, still generating sufficient cRNA for array analysis. This translates to significant savings of time and reagents.
Figure 2. ArrayScript Reverse Transcriptase Produces up to Twice the cRNA 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 70C for 10 minutes. Reverse transcription was initiated with the RT enzyme and incubated at 42C for 2 hr in an air incubator. Following first-strand cDNA synthesis, DNA polymerase was added for second-strand synthesis, which was incubated at 16C 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 cRNA was purified by glass filter and eluted in nuclease-free water for analysis by Agilent 2100 bioanalyzer and NanoDrop Spectrophotometer.
ArrayScript is Suitable for all RT Applications
Reverse transcriptases are also used in RT-PCR, cDNA library construction, and RLM-RACE. In most of these applications, synthesis of full-length cDNA is a critical parameter that affects final experimental results. For example, high yields of full-length cDNA result in the generation of cDNA libraries that more faithfully represent the cellular transcriptome. The higher yields possible with ArrayScript also make it an excellent choice for general reverse transcriptase applications that are not length sensitive, such as real-time RT-PCR.
ArrayScript is provided in two convenient sizes, 4,000 U and 10,000 U, and a 10X Reaction Buffer is provided with the enzyme.
ArrayScript is a critical component of Ambions MessageAmp II aRNA Amplification Kit. Learn how this kit outperforms other amplification products.