BioResearch
Quantitative RNA analysis with
the FlashGel™ system for RNA
By Hugh White, Lonza Rockland, Inc.
Methods
Comparison of replicate samples
Degradation time course
Introduction
Assessment of RNA quality / integrity is key to
consistent performance in a variety of uses,
including microarrays, RT-PCR, and RT-qPCR.
Chip-based analysis methods provide a fast,
sensitive, and quantitative rating of RNA quality
(RNA integrity number (RIN)). However, such
systems tend to be less flexible and more
expensive than traditional gel-based methods
of RNA analysis.
The FlashGel™ system is a rapid, ultra-sensitive,
gel-based separation system that is compatible
with image analysis packages that provide
useful quantitative information regarding RNA
quality as assessed by a ratio of the ribosomal
RNA bands.
E. coli Total RNA (Ambion) was diluted in
ultra-pure water to 40 ng/μl and subjected to
degradation by incubation at 55ºC for various
times. Following degradation, RNA samples
were prepared for analysis by combining with
an equal volume of formaldehyde sample
buffer (Lonza), followed by denaturation at
60ºC for 2 minutes. Aliquots containing 50 ng
of total RNA were analyzed by electrophoresis
on a FlashGel™ RNA cassette (225 V, 8 minutes,
20 minutes post-run hold prior to imaging).
Cassette images were captured (Syngene
ChemiGenius™) and images were analyzed using
TotalLab™ TL100 (Nonlinear Dynamics) image
analysis software.
Freshly prepared and partially degraded RNA
samples (4 days at 37ºC) were prepared and
aliquots of RNA containing 50 ng were separated
in triplicate on a FlashGel™ RNA cassette. Images
were captured using a Syngene ChemiGenius™
imaging system and the FlashGel™ camera.
Figure 1
M123456M
Table 1
55ºC Time course ribosomal band ratios
Sample
Time at 55°C
Ribosomal band radio
1
0
1.43
2
2 hours
1.47
3
4 hours
1.17
4
8 hours
0.95
5
18 hours
0.33
6
40 hours
not readable
Results
Figure 2
ChemiGenius™ imager
FlashGel™ camera
As can be seen in Figure 1, there is evidence of
increasing RNA degradation as the incubation
time is increased. The image clearly shows the
disappearance of the 23S and 16S ribosomal
RNA bands. The image was further examined to
compare the ratio of the peak areas of the 23S
and 16S ribosomal RNA bands. The data shown
in Table 1 confirms increasing RNA degradation
with increased incubation time, as illustrated by
the reduction in the ribosomal band ratio.
The images of the freshly prepared and partially
degraded RNA samples on the FlashGel™ RNA
cassette were captured using two different
systems (Figure 2). Using the TotalLab™ image
analysis software, values for ribosomal band
ratios were determined (average and standard
deviation reported; Table 2). Ribosomal band
ratios of replicate samples were also estimated
using ImageJ Version 1.41 (NIH). As can be
seen from the data presented, the ribosomal
band ratios of replicate samples were very
reproducible. The absolute values of ratios,
however, varied depending upon image capture
and image analysis methods used.
Table 2
ChemiGenius™ imager
FlashGel™ camera
Degraded
Fresh
Degraded
Fresh
TotalLab™ image analysis
average ratio ± SD
0.94 ± 0.03
1.48 ± 0.06
0.73 ± 0.03
1.39 ± 0.08
ImageJ image analysis
average ratio ± SD
0.28 ± 0.02
1.00 ± 0.03
0.38 ± 0.02
0.97 ± 0.03
Conclusion
The FlashGel™ system offers fast, ultra-sensitive
separation of RNA. Results are obtained in
approximately 30 minutes from sample loading
to image capture. Following separation, image
analysis can give semi-quantitative results for
as little as 25 – 50 ng of total RNA. Comparison
of ribosomal band ratios by image analysis
demonstrates decreasing values for samples
with increasing levels of degradation. Consistent
ribosomal band ratios were seen with multiple
methods of image capture and image analysis.
Absolute values of ratios seen were dependent
on the specific combination of methods used.
The FlashGel™ system offers the speed and
sensitivity of a chip-based analysis system in a
simpler, more flexible and less expensive format.
Lonza Rockland, Inc. – Rockland, ME 04841
For research use only.
Not for use in diagnostic procedures.
ChemiGenius is a trademark of Syngene.
TotalLab is a trademark of Non Linear Dynamics.
All other trademarks herein are marks of the Lonza
Group or its affiliates.The information contained herein
is believed to be correct and corresponds to the latest
state of scientific and technical knowledge. However, no
warranty is made, either expressed or implied, regarding
its accuracy or the results to be obtained from the use of
such information and no warranty is expressed or implied
concerning the use of these products. The buyer assumes
all risks of use and/or handling. No statement is intended
or should be construed as a recommendation to infringe
any existing patent.
© 2011 Lonza Rockland, Inc. All rights reserved.
WP-QuantRNAFlashgel 08/11
MB-WP003