What is the best RNA purification method to use to get total
RNA for the TargetAmp Kit? |
Total cellular RNA, isolated by a number of methods, including salt-fractionation
(for example using EPICENTRE's ArrayPure™ Nanoscale or MasterPure™ RNA
Purification kits), TRIzol ® / TRI Reagent and spin
columns, can be amplified successfully using the TargetAmp RNA Amplification
Kits. It is very important that the purified RNA be free of salts, metal
ions, ethanol and phenol which can inhibit the enzymatic reactions performed
in the RNA amplification process. Details of total RNA isolation methods
are presented in the TargetAmp Kit product literature that accompanies
each kit and is available on this web site. |
Can the aRNA (aminoallyl-aRNA) produced by a TargetAmp reaction
be used with either oligo arrays or cDNA arrays? |
Yes. TargetAmp reactions produce anti-sense RNA (aRNA, sometimes
called cRNA) that can be used with either oligo or cDNA arrays including
Affymetrix GeneChip®, GE CodeLink™, Agilent, and other commercial
and spotted arrays. |
Can I use the TargetAmp Kits to amplify RNA isolated from
laser-captured (or other microdissected) cells? |
Yes. The TargetAmp 2-Round Amplification Kits are the best commercial
kits for amplifying RNA from a minute number of cells. A TargetAmp 2-Round
Amplification reaction will produce microgram amounts of amplified aRNA
from the total RNA of a single cell!
See the "TargetAmp Kit Performance" web pages for more information. |
How much non-specific amplification product (background) will
I get from a TargetAmp amplification reaction?. |
Unlike other commercial kits that can produce large amounts of non-specific
amplification product, the TargetAmp kits utilize a proprietary chemistry
that virtually eliminates non-specific amplification products.
See the "TargetAmp Kit Performance" web pages for more information. |
When should I use a TargetAmp 1-Round kit and when should
I use a TargetAmp 2-Round Kit? |
The TargetAmp 1-Round Kits should be used when amplifying RNA from
25 ng to 500 ng of total RNA. Use the TargetAmp 2-Round kits when amplifying
total RNA from 1 to 50 cells (10 pg to 500 pg of total RNA).
See the "TargetAmp Selection Guide" web page for additional
information. |
I am currently performing 2-rounds of RNA amplification. Can
I get the same or better yield of aRNA (aminoallyl-aRNA) using a TargetAmp
1-Round Kit? |
In some cases, yes! A TargetAmp 1-Round Amplification reaction, which
can be performed in 6 hours, may produce more aRNA (aminoallyl-aRNA) than
some 2-round RNA amplification reactions that take up to 3 days to complete.
See the "TargetAmp Kit Performance" web pages for more information. |
EPICENTRE claims that the TargetAmp RNA amplification process
is a "simplified and improved" Eberwine RNA amplification
procedure. What have you done? |
The TargetAmp kits feature:
- Faster reaction times: A TargetAmp 1-Round Amplification reaction
can be performed in 6 hours and a TargetAmp 2-Round reaction in 2 days.
- EPICENTRE's AmpliScribe™ in vitro transcription technology
which produces the highest yield of aRNA (aminoallyl-aRNA).
- In vitro transcription reactions without the need to purify the cDNA
template.
- Optimal results using SuperScript Reverse Transcriptase enzymes (provided
by the user) which enable high temperature reverse transcription.
- Reaction components that are formulated to minimize pipetting steps.
|
Why do you recommend using SuperScript II and SuperScript III Reverse
Transcriptases in the TargetAmp process? |
We recognize that the SuperScript Reverse Transcriptase enzymes
are the most widely used RTs for gene expression studies. Researchers
trust the results obtained using these enzymes and have already generated
a significant amount of data using these enzymes. |
How long does a TargetAmp reaction take? |
A TargetAmp 1-Round Amplification reaction takes about 6 hours.
A TargetAmp 2-Round Amplification reaction can be completed in 2 days. |
Have the TargetAmp Kits been evaluated for use with Affymetrix
GeneChip® arrays? |
The aminoallyl-aRNA produced using the TargetAmp 1-Round or the
TargetAmp 2-Round Kits and subsequently labeled with biotin-NHS, yields
high quality expression profile data using Affymetrix GeneChip Arrays.
See the "TargetAmp Kit Performance" web pages for details. |
Can I biotin-label the aRNA for use on Affymetrix GeneChip® or
GE CodeLink™ arrays? |
Yes. The TargetAmp 1-Round & the TargetAmp 2-Round Aminoallyl-aRNA
Amplification Kits produce aminoallyl-aRNA which can be readily labeled
with biotin using EPICENTRE's Biotin-X-X-NHS (provided by the user).
Alternately, the TargetAmp 1-Round & 2-Round aRNA Amplification
Kits can be modified for direct incorporation of Biotin-NTP (provided
by the user) |
Can I use the TargetAmp Kits for producing Cy-labeled aRNA? |
Yes. The TargetAmp 1-Round & the TargetAmp 2-Round Aminoallyl-aRNA
Amplification Kits produce aminoallyl-aRNA which can be readily labeled
with Cy-NHS or other fluorescent-NHS dye (provided by the user).
Alternately, the TargetAmp 1-Round & the TargetAmp 2-Round aRNA
Amplification Kits can be modified for direct incorporation of Cy-NTP
(provided by the user). |
What are the advantages of producing aminoallyl-aRNA? |
The aminoallyl-method for indirect labeling of the target nucleic
acid is becoming increasingly popular because it has important advantages
over direct incorporation of a Biotin-NTP or fluorescent-NTP, including:
- The aminoallyl-aRNA produced in a single TargetAmp amplification
reaction can be divided and labeled independently with different NHS-ester
(e.g. Cy3-NHS and Cy5-NHS) if desired.
- An aminoallyl-NTP is more efficiently incorporated into the aRNA
during the in vitro transcription reaction than a biotin-NTP nucleotide
or Cy-NTP nucleotide. This results in higher yields of aminoallyl-aRNA
than can be obtained by direct incorporation of a biotin-NTP or a fluorescent-NTP.
- Conjugation of an amine-reactive biotin-NHS, Cy-NHS or other fluorescent
dye-NHS to AA-aRNA is a much less expensive way to label the target compared
to direct incorporation of a biotin-NTP, Cy-NTP or fluorescent dye-NTP.
|
Where I can get Biotin-NHS for conjugating to the aminoallyl-aRNA? |
EPICENTRE offers Biotin-X-X-NHS in convenient 2.5 mg vials for conjugating
to aminoallyl-aRNA (or aminoallyl-cDNA). A complete protocol for conjugating
to aminoallyl-aRNA is provided with the Biotin-X-X-NHS. |
What yield of aRNA (aminoallyl-aRNA) can I expect from a TargetAmp
reaction? |
A TargetAmp 1-Round Amplification reaction will produce microgram
amounts of aRNA (aminoallyl-aRNA) from as little as 25 ng of total RNA.
A TargetAmp 2-Round Amplification Kit will produce microgram amounts
of aRNA (aminoallyl-aRNA) from the total RNA isolated from as few as 1
cell (approximately 10 pg of total RNA).
See the "TargetAmp Kit Performance" web pages for more information. |
What is the size of the aRNA (aminoallyl-aRNA) produced in
a TargetAmp reaction? |
The TargetAmp 1-Round Amplification Kits produce aRNA (aminoallyl-aRNA)
with an average size of about 1200 bases as analyzed by both denaturing
agarose gel electrophoresis and the Agilent 2100 Bioanalyzer.
The TargetAmp 2-Round Amplification Kits produce aRNA (aminoallyl-aRNA)
with an average size of about 600 bases.
See the "TargetAmp Kit Performance" web pages for more information. |
1. What is the best RNA purification method to use to get
total RNA for the TargetAmp Kit? |
Total cellular RNA, isolated by a number of methods, including salt-fractionation
(for example EPICENTRE's ArrayPure™ and MasterPure™ RNA purification
kits), TRIzol® / TRI Reagent and spin columns, can
be amplified successfully using the TargetAmp RNA Amplification Kits.
However, it is very important that the purified RNA be free of salts,
metal ions, ethanol and phenol which can inhibit the enzymatic reactions
performed in the RNA amplification process. Details of total RNA isolation
methods are presented in the TargetAmp Kit product literature that accompanies
each kit and is available on this web site. |
2. When should I use a TargetAmp 1-Round kit and when should
I use a TargetAmp 2-Round Kit? |
The TargetAmp 1-Round Kits should be used when amplifying RNA from
25 ng to 500 ng of total RNA. Use the TargetAmp 2-Round kits when amplifying
from 10 pg to 500 pg of total RNA. See the TargetAmp Selection Guide web
page for additional information. |
3. Is it possible to scale up or scale down the TargetAmp RNA
amplification reaction? |
We do not recommend scaling the TargetAmp reactions up or down. The
procedure is highly optimized. Trying to get more RNA or trying to work
with low volumes may lead to sample loss or lower than expected yields. |
4. Why does EPICENTRE recommend doing the second strand cDNA
synthesis reaction at 65°C? Other commercial kits and published
procedures use 16°C. |
The TargetAmp Kits incorporate a unique, proprietary, highly-processive
thermostable DNA Polymerase to make the second strand of the cDNA, rather
than a mesophilic enzyme. |
5. Why do you recommend using SuperScript II instead of SuperScript
III during the second round of amplification in the first-strand cDNA
synthesis? |
During the second round of amplification, using high temperature
reverse transcription to achieve more full-length cDNA is not as critical
as in the first round of amplification. Using a mesophilic reverse transcriptase
such as SuperScript II generates higher yields of cDNA than with SuperScript
III. |
6. At which steps in the TargetAmp RNA amplification process
can I stop? |
The stopping points are following the 2nd-strand cDNA synthesis
steps, after DNase I treatment of the in vitro transcribed aRNA
(just prior to the aRNA purification steps) and after the aRNA purification
steps. If you choose to stop at the cDNA step you could save your sample
at -20°C. We typically store our aRNA samples at -80°C. |
7. Will there be a benefit if I increase the in vitro transcription
reaction times. |
No. The in vitro transcription reaction times are optimized
to produce the highest yield of long aRNA. Running the reactions longer
will begin to significantly reduce the NTP pool and result in shorter
aRNA products. |
8. What is the best way to purify my aRNA or aminoallyl-aRNA
after I have completed the one amplification round or two amplification
round procedures? |
The purification column to use is dependent on the expected yield
of aRNA or aminoallyl-aRNA (AA-aRNA). If the expected yield is <40 µg,
purify the aRNA or AA-aRNA using the Qiagen MinElute Cleanup Kit (Qiagen
cat. no. 74204) . If the expected yield of aRNA or AA-aRNA is >40 µg,
use the Qiagen RNeasy Mini Kit. Details are provided in the TargetAmp
Kit product literature. |
9. If I perform the in vitro transcription reactions using
a thermocycler, can I add a 4°C overnight "hold" and continue with
the aRNA purification the following day? |
Yes, this is typically what we do. |
10. In the TargetAmp2-Round amplification protocol the volume
is adjusted to 3 ul using speed vacuum just prior to the 2nd-round 1st-strand
cDNA synthesis step.
a) Can I use a MicroCon concentrator instead?
b) What will happen if I reduce the volume to less than 3 ul?
c) What will happen if I accidentally dry the reaction? |
a) We do not recommend the use of a microconcentrator system, for
two reasons. First, the molecular weight cutoff of most of the micro concentrators
is too high. It is possible to lose some of your short messenger RNAs
through the pores of the concentrator membranes, which will destroy the
accuracy of your microarray analysis using the amplified RNA. Further,
elution of concentrated RNA requires 15 µl of buffer. This volume
is too great to be used in the standard first strand cDNA synthesis reaction.
b) If the volume is dried to 1 µl to 3 µl, you should add
RNase-free water to bring it back to 3 ul.
c) The result of completely drying the aRNA sample may be lower than
expected yield of aRNA after the 2nd-round amplification because it may
be difficult to dissolve dried aRNA in 3µl of RNase-free water or
you may "miss" your aRNA when you add 3 ul of RNase-free water. |
11. The aRNA purification steps read "remove any remaining
solution from the column using a vacuum apparatus". If we do not have
a vacuum apparatus, could we use a longer centrifugation instead? |
Yes, spin it for an extra minute or two and just look to see if
the column appears to be dry, no droplets of water) in the inner surface
of the column. |
17. What is the advantage of producing aminoallyl-aRNA rather
than direct incorporation of a biotin-NTP, Cy-NTP or other fluorescent
dye-NTP into the in vitro transcription reaction? |
The aminoallyl-method for indirect labeling of the target nucleic
acid is becoming increasingly popular because it has important advantages
over direct incorporation of a biotin-NTP or fluorescent-NTP, including:
- A single RNA amplification reaction, producing aminoallyl-aRNA, can
be divided and labeled independently with different NHS-ester (e.g. Cy3-NHS
and Cy5-NHS) if desired.
- An aminoallyl-NTP is more efficiently incorporated into the aRNA
during the in vitro transcription reaction resulting in higher
yields of aminoallyl-aRNA than can be obtained by direct incorporation
of a biotin-NTP or a fluorescent-NTP.
- Conjugation of an amine-reactive biotin-NHS, Cy-NHS or other fluorescent
dye-NHS to AA-aRNA is a much less expensive way to label the target compared
to direct incorporation of a biotin-NTP, Cy-NTP or fluorescent dye-NTP.
- Inexpensive Biotin-X-X-NHS, Cy-NHS and fluorescent dye-NHS labeling
reagents are commercially available.
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