Like many other labs, we are utilizing microarray technology to gain insight into the global gene expression patterns of our protein of interest. In our lab, we are interested in studying the global role of Hap1 in gene expression in heme-deficient and heme-sufficient yeast cells. For our microarray analysis we prepared total RNA from our cells via a single-step guanidium isothiocyanate/phenol-chloroform extraction procedure. Obviously there are other methods for preparing total RNA from your cells; some would recommend isolating poly(A) RNA to detect rare mRNAs instead of using total RNA, however total RNA has worked well for us. Once the mRNA is extracted, one of the key steps is generating fluorescently labeled cDNA with either rhodamine (Cy3) or fluorescein (Cy5) which will then be hybridized onto the microarray slides. Our lab uses Ambion’s Amino Allyl cDNA Labeling Kit to reverse transcribe our RNA and to label the resulting cDNA for microarray analysis. In our hands, this kit has been efficient and consistently produces high-quality fluorophore-labeled cDNA.
Instead of directly incorporating florescently labeled dNTPs (Cy3 and Cy5-dNTPs) by reverse transcription, the Amino Allyl kit conjugates dNTPs to a reactive primary amine (amino allyl-dUTP) which is then incorporated into cDNA by reverse transcriptase. A monoreactive ester Cy3 or Cy5 dye is then coupled to the reactive primary amine. Incorporating Cy-dNTP indirectly, via the incorporation of the amino allyl-dNTP, results in a more uniform labeling of Cy3 and Cy5 labeled cDNA.
The Ambion Amino Allyl cDNA Labeling Kit comes with all the reagents required for labeling cDNA, except for the fluorescent Cy3 and Cy5 (mono-reactive NHS-esters) dyes and ethanol. We purchased our Cy3 and Cy5 dyes from Amersham Biosciences, as recommended by Ambion, but the kit is compatible with monoreactive ester Cy dyes from other companies. There are enough reagents for 15 labeling reactions and the reagents are stable for up to 6 months. Some of the reagents included in the kit are: oligo dT primers, RNase inhibitors, dNTP mix, amino allyl-dUTP, reverse transcriptase, reverse transcription buffer, and RNase free water. Also, NaOH is provided in the kit to remove template RNA after the reverse transcriptase reaction, Hepes to neutralize the reverse transcriptase reaction and sodium acetate and glycogen for the recovery of cDNA via ethanol precipitation. The kit also includes the NucAway Spin columns (and relevant tubes), coupling buffer, DMSO, and hydroxylamine for coupling the amino modified cDNA with the fluorophore dye, purifying, and concentrating the resulting fluorophore dye labeled cDNA.
Overall, we have been very pleased with this kit. It is relatively easy to use and allows for the efficient, uniform labeling of cDNA. The whole procedure can be done in half a day. Although we have not tried incorporating Cy-dNTPs directly, we have told by others that making the amino allyl cDNA (then coupling that to Cy dye) is a more effective and consistent way to produce fluorophore labeled cDNA. And the results that we have obtained with our microarray data seem to indicate that this may be true. Because of this, we have no problems recommending this product.
Hee Chul Lee
Graduate Student
Department of Biochemistry
NYU School of Medicine