1. Can you double-label aRNA with biotin-UTP and biotin-CTP?
Yes. Data in the literature indicate that the contribution of biotin-CTP to the overall signal intensity is much lower than that of biotin-UTP; however, incorporation of biotin-CTP may have some benefits for the detection of low-abundance targets. In addition to using biotin-UTP, 25%-30% of the CTP can be substituted with biotin-16-CTP without decrease of yield in one-round amplification reactions.
2. Are there any specific precautions one must take to ensure the efficiency of the aminoallyl/N-hydroxysuccinimide (NHS) coupling reaction?
Biotin-X-X-NHS is readily hydrolyzed by water and can react with nucleophilic compounds, for example the amino groups of Tris buffers. Biotin-X-X-NHS should be dissolved in dry dimethylsulfoxide (DMSO) as close to the time of use as possible. Once dissolved in DMSO or other solvent, its stability is entirely dependent on the continued absence of water or other nucleophilic compounds. Since DMSO is extremely hygroscopic and quickly takes up water vapor from the air, we recommend using biotin-X-X-NHS that has been freshly dissolved in dry DMSO. If the biotin- X-X-NHS has been dissolved and stored in DMSO, the stability of the biotin-X-X-NHS should be validated prior to using it for biotinylation of aminoallyl-aRNA from a rare or valuable sample.
3. What is the recommended ratio of UTP to biotin-UTP?
The best ratio of UTP to biotin-UTP can vary based on the nucleotide concentration and the GC content of the transcription templates. To achieve good overall signal intensity without compromising aRNA yield and length, between 25% to 40% of the UTP can be replaced with biotin-16-UTP (corresponding to ratios of UTP/ biotin-16-UTP between 3:1 and 1.5:1) in the one-round TargetAmp protocol.
4. Can I use the TargetAmp Kits for producing Cy-labeled aRNA?
Yes. The TargetAmp 1-Round and the TargetAmp 2-Round Aminoallyl-aRNA Amplification Kits produce aminoallyl- aRNA (AA-aRNA), which can be readily labeled with Cy-NHS or other types of fluorescent dye-NHS (provided by the user).
5. What are the advantages of aminoallyl-labeling over direct incorporation of a labeled-NTP?
The aminoallyl method for indirect labeling of the target nucleic acid has become increasingly popular because it has important advantages over direct incorporation of a biotin- or dye-labeled NTP. Aminoallyl-UTP is more efficiently incorporated into the aRNA during the in vitro transcription reaction than labeled nucleotides. Additionally, conjugation of an amine-reactive NHS ester of biotin (e.g., Biotin-X-X-NHS; Epicentre), 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 labeled nucleotides.
6. What are the advantages of direct labeling of aRNA?
Labeling by direct incorporation is faster. Conjugation of aminoallyl-aRNA to aminoreactive biotin- or dye-derivatives after in vitro transcription requires a 1-hour incubation, and an additional clean-up step compared to direct incorporation. Furthermore, labeling aRNA by direct incorporation does not require the use of toxic reagents (such as DMSO), which are used in the indirect labeling protocol.
7. What is the best method to clean up the labeled aRNA?
aRNA labeled by direct biotin-UTP incorporation is usually cleaned up on silica spin-columns. After coupling AA-aRNA to biotin- or dye-NHS, the labeled RNA can be cleaned up using a spin column, or with standard ion-exchange or gel-filtration methods. Another common approach is to use microconcentrators; however, microconcentrators have their own issue with sample loss (especially for shorter RNAs), so the use of silica-based spin columns is recommended.
8. Is it possible to incorporate biotin-UTP directly into the antisense RNA (aRNA) produced by the TargetAmp kits?
Yes. Epicentre has recently launched the TargetAmp 2-Round Biotin aRNA Amplification Kit 3.0, a kit that will generate amplified RNA from as little as 5 cells or 50 pg of total RNA that directly incorporates biotin-UTP into amplified RNA in the second in vitro transcription reaction. Epicentre also sells three one-round RNA amplification kits that directly incorporate biotin-UTP into the amplified RNA: the TargetAmp Biotin-aRNA Amplification Kit 104 and the new TargetAmp Biotin-aRNA Amplification Kit 105. The TargetAmp biotin-aRNA Kit 105 offers a new UTP/ biotin-UTP PreMix for optimized labeling and maximum signal intensity. It will produce microgram amounts of biotin-aRNA for use on Affymetrix® GeneChip® arrays, Illumina® BeadChips, and other microarray platforms. Epicentre also recently added the TargetAmp Nano-G Biotin aRNA Amplification Kit that uses a single round of amplification to generate aRNA for Illumina's BeadArray technologies.
9. Can the aRNA generated using TargetAmp aRNA Kits be used in next-generation RNA-Seq applications?
Yes. After aRNA synthesis, RNA may be fragmented if needed and used to create libraries using Epicentre's ScriptSeq mRNA-Seq Library Kits or any of Illumina's Tru-Seq sequencing technologies. There is no need for rRNA depletion as the TargetAmp aRNA process effectively eliminates rRNA sequences during the aRNA synthesis by amplifying only the mRNA (or other poly-A tailed RNA species). The sequencing libraries created can then be sequenced using standard techniques. As the output sequences will be those of amplified RNA that is the complement of the original RNA sample, bioinformatics techniques must me employed to "reverse complement" the sequence information; this can be easily done to all data using "PERL" Scripts with the bioinformatics software.
10. Why must I use a reverse transcriptase from a different company to perform the first round cDNA synthesis with the oligo-dT/T7 promoter primer?
Standard MMLV RT enzymes, while useful in making cDNAs in general cDNA synthesis reactions, have been found to have difficulty in being able to pick up rare transcripts from a low-mass sample input. The use of Superscript III (Invitrogen) in the first cDNA synthesis step has been found to enhance detection of such transcripts, especially critical in low mass RNA samples <1 ng). The use of such reverse transcriptases allows MessageBOOSTER to detect low-expression genes in samples with as little mass input as 10 picograms.