Over the past 10 years the MEGAscript™ Kit has earned the reputation of being the best high yield transcription kit available, and for good reason. As such, it is frequently used as a benchmark for comparison by other manufacturers who are continually trying to top the yields obtained with MEGAscript. However, yield is only half the story. A high quality transcription kit must make high yields of full-length RNA. When one compares the RNA produced by MEGAscript vs. the competition, the advantages of the MEGAscript Kit are obvious. Here we describe a set of rigorous tests comparing MEGAscript to another manufacturer's high yield transcription kit. We evaluated each kit for both yield and the capacity to synthesize full length transcripts of different lengths using three different analytical methods.
Comparing the Synthesized RNA
Following the manufacturer's instructions, 1 kb, 4 kb, and 9 kb RNA transcripts were synthesized using the MEGAscript Kit and another manufacturer's high yield transcription kit. A trace amount of [alpha-
32P]ATP was added to the reactions to facilitate accurate quantitation of RNA yield. After removing unincorporated nucleotides, RNA yield was measured by counting the trace labeled RNA in a scintillation counter and calculating yield. This method can be grossly misleading if full-length transcripts are not the only products synthesized, as it provides no information about RNA product length. For this reason, RNA quality was assessed by both formaldehyde-agarose gel electrophoresis and capillary electrophoresis on the Agilent 2100 bioanalyzer.
Similar RNA yields were produced by both kits as determined by simple mass quantitation based on TCA precipitation data (Figure 1). Examination of the RNA by bioanalyzer and gel analysis, however, reveals a very different story. Both kits were able to synthesize a 1 kb transcript, but only the MEGAscript Kit was able to produce full-length 4 kb and 9 kb transcripts. Figure 2 illustrates agarose gel analysis of the reaction products. Notice the virtual absence of full-length 4 kb and 9 kb product in the competitors' samples. Analysis on the Agilent 2100 bioanalyzer confirms and extends the gel data: full-length 4 kb and 9 kb product are barely detectable using the other kit. This startling failure prompted us to obtain a second kit of a different lot from the same manufacturer (Lots A52-90374 and A52-109100 were tested). We repeated the experiments and obtained identical results (data not shown).
While most transcription kits can synthesize a 1 kb RNA, reliable high quality synthesis of 4 kb and 9 kb RNA transcripts is a more stringent test of kit quality and reliability. Focused attention to the quality of every component and careful optimization of manufacturing processes combine to make the MEGAscript Kit a consistent and top performing product, deserving of its reputation. The next time you run across an ad for the 'best' high yield transcription kit, remember that yield is only part of the story.
Similar RNA yields were produced by both kits as determined by simple mass quantitation based on TCA precipitation data (Figure 1). Examination of the RNA by bioanalyzer and gel analysis, however, reveals a very different story. Both kits were able to synthesize a 1 kb transcript, but only the MEGAscript Kit was able to produce full-length 4 kb and 9 kb transcripts. Figure 2 illustrates agarose gel analysis of the reaction products. Notice the virtual absence of full-length 4 kb and 9 kb product in the competitors' samples. Analysis on the Agilent 2100 bioanalyzer confirms and extends the gel data: full-length 4 kb and 9 kb product are barely detectable using the other kit. This startling failure prompted us to obtain a second kit of a different lot from the same manufacturer (Lots A52-90374 and A52-109100 were tested). We repeated the experiments and obtained identical results (data not shown).
While most transcription kits can synthesize a 1 kb RNA, reliable high quality synthesis of 4 kb and 9 kb RNA transcripts is a more stringent test of kit quality and reliability. Focused attention to the quality of every component and careful optimization of manufacturing processes combine to make the MEGAscript Kit a consistent and top performing product, deserving of its reputation. The next time you run across an ad for the 'best' high yield transcription kit, remember that yield is only part of the story.
Figure 1. Mass Amounts of RNA Transcripts Produced by Two Distinct Transcription Systems. 1 kb, 4 kb or 9 kb templates were used in Ambion's MEGAscript Kit or another manufacturer's in vitro transcription kit according to protocol. 1 µl of each [alpha-32P]ATP-labeled reaction was suspended in 200 µl of carrier DNA and split into equal aliquots. One aliquot was counted directly in a scintillation counter and the other was precipitated with 10% cold TCA and filtered, before counting.
Figure 2. RNA Products From Two Transcription Systems. 1 kb, 4 kb or 9 kb templates were used in Ambion's MEGAscript™ Kit or in another manufacturer's in vitro transcription kit according to protocol. A portion of each reaction (5 µl) was loaded onto a denaturing 1% agarose gel and stained with ethidium bromide.
Figure 3. Electropherogram of RNA Transcripts of Different Sizes From Two Transcription Systems. 1 kb, 4 kb or 9 kb templates were used in Ambion's MEGAscript™ Kit or in another manufacturer's in vitro transcription kit according to protocol. 20 µl reactions were diluted to 100 µl, and 1 µl was assessed on the Agilent 2100 bioanalyzer. The single primary peak is indicative of product quality while the area under the peak is related to yield.