For the past several months I have been using in-house fabricated cDNA microarrays to study the differential gene expression of approximately 100 human genes. To do this, I have been incorporating fluorescent nucleotide analogues into test or reference RNAs using reverse transcriptase (i.e. Cy3-dCTP and Cy5-dCTP respectively). During the course of my experiments, I found that the critical parameters for the efficient labeling of cDNA include: 1) the amount of starting mRNA 2) the priming method used (i.e.oligo(dT) vs. random priming) 3) the concentration of ‘hot’ and ‘cold’ nucleotides (i.e CyDye labeled or unlabeled nucleotide analogues, respectively) and 4) the amount of reverse transcriptase used. When I first started these experiments, it took quite an effort to optimize all these parameters, but overall I had success with this method. The major drawback used to be the relatively low incorporation efficiency of the bulky CyDye molecules.
For the past several months I have been using in-house fabricated cDNA microarrays to study the differential gene expression of approximately 100 human genes. To do this, I have been incorporating fluorescent nucleotide analogues into test or reference RNAs using reverse transcriptase (i.e. Cy3-dCTP and Cy5-dCTP respectively). During the course of my experiments, I found that the critical parameters for the efficient labeling of cDNA include: 1) the amount of starting mRNA 2) the priming method used (i.e.oligo(dT) vs. random priming) 3) the concentration of ‘hot’ and ‘cold’ nucleotides (i.e CyDye labeled or unlabeled nucleotide analogues, respectively) and 4) the amount of reverse transcriptase used. When I first started these experiments, it took quite an effort to optimize all these parameters, but overall I had success with this method. The major drawback used to be the relatively low incorporation efficiency of the bulky CyDye molecules.
About 3 weeks ago I came across GE Healthcare' Cyscribe kit to prepare CyDye labeled cDNAs from mRNA for microarray hybridization. I can highly recommend this kit to anybody new in the microarray field who does not want to spend the long hours it takes to establish a new protocol. In my hands, this kit works well with as low as 0.1 ug mRNA and gives hybridization signals about 5-fold greater than the protocol I used to follow. GE Healthcare claims that the higher labeling efficiency of their system is based on point mutations introduced in the CyScript reverse transcriptase, making it more tolerable to cy3- or cy5 modified nucleotides. The kit also comes with a formamide based microarray hybridization buffer that I have been using for overnight hybridizations. By including poly(dA) and Cot-1 DNA to block non-specific hybridizations I see consistently low background.
Three alternative priming methods are available with this kit: Priming with anchored oligo(dT) will direct the start of the synthesis of cDNA from the 5’ end of the polyA-tail. This priming method is especially suitable if the hybridization targets on the microarray are derived from the 3’ ends of transcripts, however, cDNA synthesis can also be primed with random nonamers. In the ‘standard’ protocol 0.1-1ug mRNA is primed using a mix of oligo(dT) primer and random nonamers (which come with the kit). The labeling reaction, catalyzed by CyScript reverse transcriptase incorporates Cy3-dCTP, Cy5-dCTP, Cy3-dUTP or Cy5-dUTP into first-strand cDNA. Two optimized nucleotide mixes are provided to be used with CyDye-dCTP or CyDye-dUTP. Labeled cDNA needs to be purified from unincorporated nucleotides and mRNA template to allow for high quality hybridizations. Excess RNA is degraded by alkaline hydrolysis followed by removal of protein and residual nucleotides.
The Cyscribe kit offers an optimized system that allows for efficient labeling of cDNA without purchase of additional components and laborious optimization experiments. Purification steps can be performed as described in the protocol with GE Healthcare AutoSeq G-50 spin columns. As outlined in the user’s manual, this kit is set up for use with mRNA, however, some investigators might prefer to use total RNA as starting material. In my hands, experiments carried out with total RNA using the Cyscribe kit did not give improved sensitivity.
Overall, this kit combines high sensitivity with short ‘hands on’ time making it ideal for high throughput screening. The easy and fast use of this kit makes it a state-of-the-art protocol for microarray based gene expression analyses.
Martin Bilban, MSc
Graduate Student
Department of Cell Biology
The Scripps Research Institute