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. |
