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Tips from the Bench
RNA Amplification:
Stop and Start Your Reactions without Affecting aRNA
Size and Yield
MessageAmp™ II aRNA Amplification
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Figure 1. Schematic Diagram of MessageAmp™ II Protocol. Two additional stopping points are indicated in red. Please refer to the Instruction Manual for complete details. |
Synthesis of amplified antisense RNA (aRNA or cRNA) is a multistep process requiring several enzymatic reactions (Figure 1). Researchers frequently inquire if there are stopping points during the RNA amplification procedure that will not compromise aRNA quality. Here we look at two steps at which the procedure can be temporarily suspended without compromising either aRNA size or yield.
Stopping After First Strand Synthesis
To determine whether it is possible to suspend the RNA amplification procedure after first strand cDNA synthesis, we compared aRNA generated from the MessageAmp™ II aRNA Amplification Kit (patent pending) using two experimental conditions. After completion of first strand cDNA synthesis, reactions were either frozen and thawed before second strand cDNA synthesis or used immediately for second strand cDNA synthesis. Inputs of both 100 and 1000 ng total RNA were tested in triplicate. Figure 2 Panel A demonstrates that aRNA yields were similar across each replicate for both amounts of starting total RNA irrespective of whether the first strand cDNA had been frozen or not. Agilent® 2100 bioanalyzer traces of the aRNA reveal that the size distribution of aRNA was also not affected (Figure 2 Panel B). Thus, we conclude that storing the first strand product at -20°C prior to second strand synthesis does not appear to affect final aRNA yield or length.
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Figure 2. First
Strand cDNA Can Be Frozen without Affecting aRNA Yield
or Length. (A) Triplicate
MessageAmp™ II reactions with either
100 (left) or 1000 (right) ng total RNA input were
compared. Half of the reactions (green, control) were
performed as instructed--the procedure was stopped
after the second strand synthesis, and a 4 hr in vitro
transcription (IVT) reaction was performed the next
day. Half of the reactions (blue, Interrupted Rxn)
were stopped earlier (after the first strand synthesis)
before completing the aRNA procedure. Yields, as measured
on a NanoDrop® ND-1000A Spectrophotometer,
indicate that stopping and freezing the aRNA synthesis
procedure after first strand synthesis does not affect
aRNA yield for a given amount of input RNA. (B) Representative
samples from Panel A were analyzed on an Agilent® 2100
bioanalyzer to show that the different reaction conditions
also did not affect aRNA size.
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Stopping and Restarting the IVT Step
The in vitro transcription step requires a relatively long incubation (4-16 hr) to maximize aRNA length and yield. We examined whether this reaction could be stopped and restarted without affecting these aRNA parameters. aRNA was synthesized from replicate 100 and 500 ng total RNA samples with the MessageAmp II Kit using either a 4 hr in vitro transcription (IVT) reaction (4 hr control), or a 2 hr reaction that was followed by freezing the reaction overnight, thawing, and continuing the reaction for an additional 2 hr (2+2 hr). For both inputs, yields were essentially identical regardless of whether the in vitro transcription reaction was stopped and restarted (Figure 3 Panel A). Agilent bioanalyzer analysis of the resulting aRNA showed that all of the traces for all samples overlapped. The only difference is that more aRNA was synthesized from samples that were generated from 500 ng total RNA inputs regardless of whether or not they were stopped and resarted (Figure 3 Panel B). Thus, stopping and restarting the in vitro transcription step also does not appear to affect aRNA yield or length.
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Figure 3. The
In Vitro Transcription Step Can Be Interrupted without
Affecting aRNA Yield or Length. (A) Replicate
MessageAmp™ II reactions with either 100 (left)
or 500 (right) ng total RNA input were compared. Half
of the reactions (green, 4 hr control) were performed
as instructed--the procedure was stopped after the
second strand synthesis and a 4 hr in vitro transcription
(IVT) reaction was performed the next day. Half of
the reactions (blue, 2+2 hr) were frozen in the middle
of the IVT reaction before completion of the aRNA procedure.
Yields, as measured on a NanoDrop® ND-1000A Spectrophotometer,
indicate that stopping and freezing the reaction during
the IVT step does not affect aRNA yield for a given
amount of input RNA. (B) Samples from Panel
A were analyzed on an Agilent® 2100 bioanalyzer
to show that the different reaction conditions also
did not affect aRNA size. |
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