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SuperScript™ Indirect cDNA Labeling System - FAQs

查看更多产品信息 SuperScript™ Indirect cDNA Labeling System - FAQs (L101402)

4 个常见问题解答

Can the amino-labeled DNA generated with your SuperScript Indirect Labeling kit be ethanol precipitated and stored overnight? Do you know if the amino-labeled DNA will be stable over that time?

Yes, the amino-labeled cDNA should be stable if stored precipitated in ethanol at -20 degrees C.

What is the ratio of modified/unmodified dNTPs in your SuperScript Indirect Labeling Kit?

The ratio of nucleotides is one of the key features in the optimized performance of this product, and therefore is considered proprietary. Unfortunately, we are unable to provide this information.

Why are there only two amino-modified nucleotides in the dNTP mix for cDNA indirect labeling (aminoallyl-dUTP and aminohexyl-dATP) intead of all four nucleotides being modified? Would more modified nucleotides have an adverse effect on the microarray?

Using four modified nucleotides rather than two can cause fluorescence quenching since the fluorophores are positioned very closely to one another. Use of two amino-modified nucleotides in the cDNA synthesis reaction (rather than one) results in a greater incorporation of fluorescent dye and higher signal intensity with small amounts of RNA starting material. Unbiased incorporation of amino-modified dNTPs and the high efficiency of the coupling reaction result in an even distribution of fluorescent signal and high overall levels of fluorescence, increasing the sensitivity and reproducibility of array hybridizations. In addition to quenching, a very high dye density on the cDNA will interfere with hybridization and, therefore, yield lower microarray fluorescence signals. We found that the use of 2 amino-modified nucleotides (at the appropriate concentration) results in optimal incorporation and high microarray signals.

Can I use the SuperScript Indirect cDNA Labeling System for bacterial samples?

We have not used bacterial RNA for the validation of the SuperScript Indirect cDNA Labeling System, however the kit should work reasonably well with few modifications to the protocol.

For total RNA samples, Oligo(dT)20 primers should be used. As the manual suggests, random hexamers should be used only with mRNA or with the RNA ladder, which is included as a control. However, in the case of bacterial RNA, longer random primers should be used instead of the random hexamers included in the kit. 18mers should work well, although the temperature of reverse transcription may need to be slightly adjusted in order to obtain optimal cDNA synthesis. The reason for using longer random primers is that the rRNA in bacterial samples will also be used as template for reverse transcription, and the target cDNA will be more complex than the corresponding cDNA primed with the Oligo(dT)20. Therefore, when doing hybridizations, you can expect some degree of cross-hybridization to occur. Nevertheless, the use of long random 18mers is common practice among microarray users.