The TrueLabeling-AMP™ 2.0 Kit from SuperArray Biosciences is used to make biotin-labeled cRNA target probes from total RNA; labeled probes are then used for hybridization to oligo arrays. As stated by SuperArray Biosciences, this kit is the “one-tube, 2 ½ hour linear amplification and labeling of cRNA target for Oligo GE Array® Hybridization”. Biotin-labeled cRNA target is synthesized from total RNA in 3 easy steps: 1) cDNA synthesis from total RNA 2)
in vitro transcription of cDNA to labeled cRNA, and 3) cRNA clean-up by column purification. The cRNA probes are then used for hybridization to SuperArray’s oligo (gene) arrays; array spot intensities are compared between groups of arrays to determine differential gene expression including fold changes in expression.
Our labs used this cRNA labeling kit to label over 40 target probes made from both rat heart and lung total RNA. We used one microgram of total RNA in the cRNA synthesis reaction. Synthesizing the cRNA probes was straightforward and could be done easily in one PCR tube on a thermocycler; however, the efficiency of biotin-labeling and cRNA synthesis with this kit was quite low and often multiple reactions were needed to generate enough cRNA for array hybridizations. We could see this when checking the cRNA before and after column purification because cRNA yields were drastically reduced as most nucleotides were not incorporated into the final cRNA product. Typically, in our labs the kit had less than a 50% success rate, meaning that little or no cRNA product was synthesized in most initial reactions. Reactions were repeated two or more times, usually with longer incubation times, more biotin or more total RNA added to the reaction in order to generate enough cRNA probe for array hybridizations. This caused us to use much more of the biotin and sample in the labeling reaction than expected and the cRNA probes became quite expensive and time-consuming to make –
Not to mention the loss of precious RNA sample! Finally, we ended up using the minimum amount of probe necessary for hybridization to 10 arrays (a minimum was 2 micrograms cRNA as suggested by one of SuperArray’s manuals- this was the most we could get for some samples anyway!).
In addition, the 1.5 kit was inconveniently upgraded in the midst of our experiments and all probes needed to be resynthesized with the newer 2.0 version in order to make valid comparisons. As anyone working with RNA knows, this kind of unforeseen sample loss and need for repetition is very frustrating. In addition, the user manuals and kit performance were inconsistent and often confusing (e.g., different labeling of reagents between kits, inconsistent directions between kits, and what to expect in terms of yields, 260/280 ratios, etc.). But we were quite satisfied with the technical feedback and advice from SuperArray’s customer service department. In the end, however, our array experiments ended up being quite costly and yet did not give as good of a result as expected.