RNA Amplification
Tips:
Tip 1:
Coprecipitants Improve RNA Recovery Without
Affecting Amplification
A detailed study by Q. Tian Wang et
al (1) addresses the effect that coprecipitants have on linear
amplification of RNA. Coprecipitants
are often used during RNA isolation to increase RNA recovery
during precipitation. This analysis was carried out using 10
ng of yeast total RNA precipitated in the presence of increasing
amounts of linear acrylamide (5 to 25 ng) or using a combination
of linear acrylamide (5 ng) and increasing amounts of yeast tRNA
(50-500 ng) as carriers. The precipitated RNA was then converted
into cDNA and amplified by linear amplification. The use of a
combination of linear acrylamide and tRNA improved the total
RNA recovery. Inhibitory effects of tRNA on cDNA synthesis were
not observed in this study, and aRNA product sizes and yields
obtained after amplification were indistinguishable from products
obtained in the absence of carriers. Experiments in Ambion's
own laboratories corroborate this result. Analysis of the aRNA
probes prepared from RNA samples containing 0, 50, and 250 ng
of yeast tRNA carrier on an Affymetrix GeneChip® system yielded
average "r" values of 0.991 0.994 and 0.995, respectively. Thus
the use of yeast tRNA supplemented with linear polyacrylamide
allowed efficient cDNA synthesis and amplification of aRNA from
nanogram quantities of starting total RNA.
Ambion offers a variety of high
quality coprecipitants. These include Glycogen, GlycoBlue™ (glycogen
derivatized with a blue dye to increase pellet visibility),
sheared Yeast Total RNA,
Yeast tRNA, and Linear Acrylamide. Each coprecipitant is subjected
to stringent quality
control tests to ensure that it is free of RNases as well as other unwanted
contaminants.
Tip 2: Double Stranded cDNA Clean-up
Different
linear amplification protocols call for different double stranded
cDNA purification methods
prior to amplification.
This step is designed to purify the cDNA template from free nucleotides,
oligo(dT)-T7 primers, and salts. A recent publication by Zhao
et al (2) found that gel filtration based purification of double
stranded cDNA followed by phenol chloroform extraction resulted
in decreased yields of aRNA due to loss of the double stranded
cDNA. At Ambion, we use a column binding and elution method that
allows elution of the double stranded cDNA in small volumes.
In addition to avoiding the need to phenol extract, this eliminates
extra vacuum drying or precipitation steps to concentrate the
cDNA before in vitro transcription and results in up to 2X higher
aRNA yields compared to phenol based purification (Figure 1).
The column binding method is available as the DNAclear™ Kit for
the purification of cDNA templates for subsequent aRNA amplification
and is included in Ambion's MessageAmp
Kits.
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|
| Figure 1. Comparison
of Two cDNA Purification Methods for Subsequent aRNA
Amplification. Two
identical rat thymus total RNA (2 µg) samples
were amplified using the MessageAmp™ aRNA
Amplification Kit. During the amplification protocol,
the double stranded cDNA from one of the samples was
purified using phenol:chloroform: isoamyl alcohol (pH
7.9) whereas cDNA from the other sample was subjected
to Ambion's DNAclear™ (column
based) clean-up protocol. |
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Ordering Information for Ambion Products:
|
| Cat# |
Product Name |
Size |
| AM1750 |
MessageAmp™ aRNA Kit |
20 rxns |
| AM1752 |
Amino Allyl MessageAmp™ aRNA Kit |
20 rxns |
| AM1756 |
DNAclear™ Kit |
20 purifications |
| For Research Use Only. Not for use in diagnostic procedures. |
|
| TechNotes
Archive |
| Ordering
Information |
References
1. Wang QT, Xiao W, Mindrinos
M, and Davis RW. (2002) Yeast tRNA as carrier in the isolation
of microscale RNA for global amplification and expression profiling. BioTechniques 33:
788-96.
2. Zhao H, Hastie T, Whitfield
ML, Borresen-Dale A-L, Jeffrey SS. (2002) Optimization and
evaluation of T7 based RNA linear amplification protocols for
cDNA microarray analysis. BMC Genomics 3: 31. |
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Analysis of Microarray Data
[read]
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Array Analysis Step by Step: Ambion's Array Analysis Products
[read]
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Array Analysis: The Basics
[read]
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Microarray Analysis: Gene Representation in Amplified vs. Unamplified RNA
[read]
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RNA Interviews: James Eberwine
[read]
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