TargetAmp™: Selection Guide | Process | Performance | FAQ | Poster (3 MB PDF)
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.
Yes, this is typically what we do.
The purification column to use is dependent on the expected yield of aRNA or aminoallyl-aRNA (AA-aRNA).
The stopping points are the following:
See TargetAmp™ aRNA Amplification Performance for more information.
The reagents have high concentrations and will precipitate out of solution when cold and they are hard to get back into solution. This will cause incorrect concentration in the reaction and yields will be low. Please make sure to warm all reagents up to room temperature before use including the enzyme.
No. The microconcentrators remove small RNAs from the sample, which will destroy the accuracy of your microarray. In addition the elution volume is 15 µl, which is too high.
If the volume is dried to less than 1 µl, you should add RNase-free water to bring it back to 3 µl.
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 3 µl of RNase-free water or you may "miss" your aRNA when you add 3 µl of RNase-free water.
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.
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.
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.
We recommend using Epicentre's ArrayPure™ and MasterPure™ RNA purification kits or another method that yields RNAs that are free of salts, metal ions etc. that can inhibit downstream enzymatic reactions. For more information on RNA isolation methods please refer to the protocol.
High temperature reverse transcription is possible using SuperScript III which allows for better sensitivity and full length cDNA synthesis. This is crucial for being able to capture rare low expressing transcripts and longer mRNAs in a TargetAmp library. SuperScript II is used in the second round, when the bigger concern is the yield of cDNA and in vitro transcription. To date we have not found any other combination of reverse transcriptases that can provide full length cDNAs, high yields and retention of rare transcripts.
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 RNA from the total RNA of a single cell!
See the TargetAmp Selection Guide for additional information.
Both the 1-Round and 2-Round kits produce microgram amounts of aRNA (aminoallyl-aRNA).
Yes. There is a paper from the lab of Dr. Steven Potter at Cincinnati Childrens Hospital (Dev Biol. 2006 May 15;293: 540-54) and from Dr. James Eberwine University of Pennsylvania (Schubert, C. Nature 480: 133-137 [December 2011]).
The yields are based on the mass of input RNA being used in the amplification reaction. Using a one-round RNA amplification on a sample size that is better suited for the TargetAmp two-round kits will not improve your yields. If your input RNA is less than 25 ng, you must use the TargetAmp two-round aRNA kits and reduce the mass of input RNA to no more than 500 pg. If the input mass of total RNA for amplification is greater than 25 ng, the TargetAmp one-round kit can be used.
Yes. There are two ways to be able to do this:
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.
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.
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: