I have been using in-house fabricated cDNA microarrays to study differential gene expression of approximately 150 human genes using incorporation of fluorescent nucleotide analogues by reverse transcription to label test or reference RNA (i.e. Cy3-dCTP and Cy5-dCTP, respectively). In my experience, I have found that the crucial parameters for the efficient labeling of RNA include finding the optimum concentrations of ‘hot’ and ‘cold’ nucleotides (i.e CyDye labeled or unlabeled nucleotide analogues, respectively), optimizing the priming method, and also finding the right reverse transcriptase (RT). This last issue is especially critical because it is important that the RT being used can incorporate the bulky CyDye-modified nucleotides efficiently and with high specificity. Early in my studies, one of the major drawbacks used to be the relatively low incorporation efficiency of the bulky CyDye labels. Some time ago I came across Qiagen’s OmniScript Reverse Transcriptase system to prepare CyDye labeled cDNA from total RNA (or mRNA) for microarray hybridization. Since then, I have used this enzyme over a wide range of total RNA concentrations and have come to the conclusion that this system really works. In my hands, overall signals on our customized microarrays improved by approximately 2-5 fold, which corresponded well with what Qiagen promises in their technical bulletin.
Qiagen claims that the higher labeling efficiency of their system is based on their distinct RT, which is different from either Moloney murine leukaemia virus (MMLV) or avian myeloblastosis virus (AMV) reverse transcriptase. Qiagen offers 2 protocols for incorporation of Cy3/Cy5-dCTPs or dUTPs from either 50ng - 2µg (protocol 1) or 5 – 50 µg RNA (protocol 2). In both protocols, cDNA synthesis is primed by oligo(dT). This priming method is especially suitable for microarray analysis if the hybridization targets on the microarray are derived from the 3’ ends of transcripts. However, if this is not the case, the kit also comes with instructions for cDNA synthesis using random primers. Labeled cDNA needs to be purified from unincorporated nucleotides and mRNA template to allow for high quality hybridizations. To do this, the samples are digested by RNase treatment followed by passing the fluorescent cDNA over Qiagen’s PCR purification column to remove unincorporated nucleotides.
There are definitely features of this kit that make it useful for microarray analysis. Probably the most important being that the OmniScript RT allows for the labeling of even very small amounts of RNA (i.e. less than 500 ng total RNA). It is also nice that the purification procedure (after labeling) is simple. The purification steps can be performed as described in the protocol using Qiagen’s PCR purification kit. The main drawback to this kit is that it does not provide other reagents needed for the preparation of labeled cDNA, such as nucleotides and primers. Also, it may show some non-specific products when using large amounts of starting total RNA.
Overall, my experience with Qiagen’s OmniScript RT Kit has been good. It allows for the efficient labeling of a wide range of starting RNA concentrations and increased the signal of our microarrays by 2-5 fold.
Martin Bilban, MSc
PhD Student
Quaranta Lab
Department of Cell Biology
The Scripps Research Institute