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.
|
