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Introduction:
DNA microarray technologies are powerful tools capable of
measuring the expression profile of many genes simultaneously. Early
microarray protocols have called for the use of RNA as a template in a
reverse transcription reaction to synthesize and label first strand cDNA
target, which is then hybridized with the DNA microarray. However in many
research applications, the limited amount of RNA sample available is usually
not sufficient for this protocol. One of the most popular techniques employed
to overcome this limitation is in vitro transcription (IVT)-based RNA
amplification. This technique lowers the minimal requirement for total RNA to
100 ng from the microgram amounts needed for conventional reverse
transcription. However, the traditional IVT method involves several reactions
and intermediate clean-up steps, and the entire procedure usually takes
a-day-and-a-half to complete making it both expensive and time-consuming. A
new IVT-based RNA amplification and labeling technique achieves a balance
between performance, convenience and cost-effectiveness. The TrueLabeling-AMP™
Linear RNA Amplification Kit is easier to use and less
expensive than the traditional method yet offers comparable performance.
1. The TrueLabeling-AMP Method:
Figure 1 shows a schematic diagram of the TrueLabeling-AMP RNA Linear
Amplification protocol. The entire procedure takes place in only one tube and
within 5.5 hours while still delivering robust RNA amplification performance.
Only 0.1 to 3 µg of total RNA or 50-100 ng of mRNA is needed for starting
material. The first step of the TrueLabeling-AMP procedure is the synthesis
of cDNA, or the reverse transcription of RNA. In the second step, a four-hour
IVT reaction is performed during which complementary RNA (cRNA) transcript
target labeled with biotinylated UTP is generated. Finally, labeled cRNA
target is purified using standard spin columns. The final purified cRNA is
ready for hybridization with cDNA- or oligo-based microarrays only 5.5 hours
after starting the reaction. Further comparisons between the key features of
the traditional and TrueLabeling-AMP IVT methods are summarized in Table 1.
| Table 1: Comparison of Features
Between TrueLabeling-AMP and Traditional IVT |
| |
TrueLabeling-Amp™ |
Traditional
|
| Input Total RNA |
0.1 - 3 µg
|
1.0 - 10 µg
|
| Typical yield of cRNA(from 3 µg total RNA) |
30 - 40
µg
|
40 - 50 µg
|
|
Labeled Product
|
Linear amplified cRNA with 3'-bias
|
Linear amplified cRNAwith 3'-bias
|
| Time Consideration 5.5 hours 1.5 to 2 days |
5.5 hours
|
1.5 to 2 days
|
| Procedure |
Single vial, 2 step reaction
|
Multi-step procedure
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| Cost Per Reaction |
< $30
|
>$65
|
|
|
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Figure 1. Schematic representation of the TrueLabeling-Amp™
Linear RNA Amplification protocol for the generation of amplified and
labeled target for microarray analysis
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2. Comparing Gene Expression Profiles Generated Using the Two IVT Methods:
The protocol for the traditional IVT method requires first
strand cDNA synthesis, RNase treatment, second strand cDNA synthesis, an
intermediate purification step, an overnight IVT, and finally a spin-column
purification step to obtain labeled cRNA product. To validate the new more
convenient method, gene expression profiles generated from both the
traditional IVT method and the TrueLabeling-AMP method were compared using an
Oligo GEArray® from SABiosciences. Figure 2 shows a high degree of
correlation between the gene expression profiles obtained by the shorter and
the longer procedures. This result demonstrates that the simple half-day
TrueLabeling-AMP protocol delivers microarray analysis performance as
reliable as the traditional IVT method.
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| Figure 2. The TrueLabeling-AMP protocol generates gene expression
profiles comparable to the traditional IVT method. Equal amounts (1
µg) of XpressRef™Human Universal Reference Total RNA (GA-004) were
used to generate amplified and labeled cRNA target using either the
TrueLabeling-AMP™Linear RNA Amplification Kit (GA-010) or a
traditional method of IVT. Equal amounts (4 µg) of cRNA were then
hybridized to the Oligo GEArray® Human Cancer Microarray (OHS-802). The
raw intensity values of each of the 440 genes on both arrays were
plotted against each other, and the data were fit to a straight line
with a correlation factor of 0.9729.
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3. Comparing TrueLabeling-AMP Expression Profiles Obtained Under
Different Conditions:
An important characteristic of a reliable RNA amplification method is the capability to deliver similar amplification performance when using various amounts of input RNA sample and when using either total or messenger RNA. Figure 3 shows that comparable gene expression profiles can be generated from RNA amounts as different as 500 ng and 3
µg. Figure 4 displays a representative comparison and a strong correlation between expression profiles using cRNA target generated by the TrueLabeling-AMP method from either total or messenger RNA. When using mRNA as starting material for this method, an excellent signal-to-noise ratio and a high percentage of positive calls were also observed (data not shown). Therefore, the TrueLabeling-AMP method is very versatile and adaptable to different amounts and types of starting material.
Figure 3. The TrueLabeling-AMP protocol generates similar gene expression
profiles independent of the amount of input RNA. Two different amounts
(500 ng or 3 µg) of XpressRef™ Human Universal Reference Total RNA (GA-004)
were used to generate amplified and labeled cRNA target with the TrueLabeling-AMP™Linear RNA Amplification Kit (GA-010). Equal amounts of cRNA
target were then hybridized to the Oligo GEArray® Human Cancer Microarray
(OHS-802). The raw intensity values of each of the 440 genes on both arrays
were plotted against each other, and the data were fit to a straight line
with a correlation factor of 0.9775.
Summary:
The TrueLabeling-AMP Linear RNA Amplification Kit method is a simple, quick,
and cost-effective way to obtain amplified and labeled cRNA target for
microarray analysis. The method is easy to use requiring only a one-vial
protocol and less than a day to finish. The simplified protocol requires
fewer reagents and purification steps lowering the cost to a fraction of
traditional IVT method. Despite these improvements in time and cost, the
method still delivers robust gene expression profiles comparable to more
traditional IVT methods. The appearance of those profiles is also independent
of the amount or type of RNA starting material used. Therefore, successful
microarray experiments can be performed in a manner that overcomes the
shortcomings of traditional IVT methods with both time saving and cost saving
benefits. The experiments shown here utilize the Oligo GEArray® from
SABiosciences. The use of this method with other microarray platforms
has not been tested; however, the performance of TrueLabeling-AMP in such
cases is expected to be similar.
Figure 4. The TrueLabeling-AMP protocol generates similar gene
expression profiles independent of the type of input RNA. Either
XpressRef™ Human Universal Reference Total RNA (3 µg) or mRNA (0.3 µg)
purified from the same total RNA sample using the ArrayGrade mRNA
Purification Kit (GA-002) was used to generate amplified and labeled cRNA
target with the TrueLabeling-AMP™ Linear RNA Amplification Kit (GA-010).
The cRNA target (8 µg from total RNA or 4 µg from mRNA) was then hybridized
to the Oligo GEArray® Human Cancer Microarray (OHS-802). The raw intensity
values of each of the 440 genes on both arrays were plotted against each
other, and the data were fit to a straight line with a correlation factor of
0.9915.
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