Labeling Reverse Transcription

After isolation and purification, total RNA is reverse-transcribed to cDNA, at which point it is labeled in a process known as "direct labeling" [30]. cDNA is a much more convenient and stable molecule to work with than mRNA. This is because RNA has a very short half life and is very easily degraded by ubiquitous RNases. Approximately 2-10 ig of bacterial RNA are used from each sample in a microarray experiment. During the reverse transcription, labels are incorporated into the cDNA. These are usually dCTP molecules labeled with the fluorescent markers Cy3 or Cy5, which fluoresce at different wavelengths. In a type 1 experiment the control and experimental RNA populations must be labeled differently, whereas in a type 2 experiment both the control and experimental RNA populations are labeled using the same fluorescent dye whilst the gDNA standard is labeled using the other.

Because microbial mRNAs are not polyadenylated, this feature cannot be targeted with primers during the reverse transcription. Therefore, the reverse transcription of a bacterial total mRNA population requires another approach. Using random primers was the first method developed, but, as Talaat et al. [31] have shown, these are often insensitive, and their nonspecificity results in some mRNAs not being transcribed at all. This can be avoided if genome-directed primers are used. These are designed by an algorithm that has been written to calculate the minimum number of primers needed to amplify every ORF in a genome. This ensures that the primers are highly sensitive to all mRNAs in a cell. Using this algorithm, it has been shown that a set of just 37 oligonucleotide primers is enough to amplify every ORF (approximately 4000) in the M. tuberculosis genome, and that they were far more specific when it came to selectively amplifying mycobacterial, rather than mammalian, RNA [31]. If gDNA is being used, as in a type 2 experiment, it is labeled using random or genome-directed primers and the DNA polymerase I Klenow fragment.

The reverse transcription process used to convert the mRNA population into cDNA prior to hybridization is not 100% efficient [32]. The 3' end of a gene is often over-represented as occasionally reverse transcriptase only synthesizes a short sequence before falling off. A direct-labeling approach where the mRNA population is labeled without the need for a reverse transcription or amplification step has been developed [33]. Although not widely used, this method promises to reduce bias and increase the accuracy of microarray results.

There are also methods of RNA labeling that improve the efficiency of the labeling reaction and are designed for small (submicrogram) yields of RNA; this is known as "cold labeling" [34]. This is necessary, for example, when extracting bacterial RNA from in vivo experiments because bacteria in the host are then usually far more diffuse than when grown in vitro. This protocol first reverse transcribes the mRNA to cDNA using unlabeled nucleotides. The cDNA is then randomly primed and labeled using the Klenow fragment of DNA polymerase, which has a much higher rate of label incorporation than does reverse transcriptase [35]. This method therefore allows either smaller amounts of RNA to be used (reportedly as little as 0.35 ig of RNA) or, in principle, lower concentrations of Cy dyes, which would make microarray experiments much more cost-effective.

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