The AmpoLabeling-LPR Kit is designed to enzymatically convert experimental RNA into labeled cDNA target for hybridization to the cDNA GEArray from SuperArray. The system is based on a linear polymerase replication (LPR) and labeling of only target genes as per the cDNA GEArray to be used.
In our lab, we normally use this labeling system to prepare probes from the RNA of endothelial cultured cells (both primary and cell lines) for hybridization of pathway specific cDNA arrays. For the RNA extraction, we use the RNeasy Kit from Qiagen, which we have been using over the years. On average, we used 500 ng of total RNA for the labeling reaction (although the manufacturer says that it can be used with ¡§as little as 100 ng¡¨) together with biotin-16-dUTP (Roche). This generates a decent amount of labeled probe to use in the hybridization. The system is easy to use as all steps can be performed in a PCR machine. In brief, the kit includes all the nucleotides, buffers and polymerases required; only the RNA and the biotin need to be added. However, there is a particular reagent which is specific for each array and is supplied by the company together with the cDNA Array Kit, but independently of the labeling kit. As the kit works, one only needs to put the reagents with the RNA in eppendorf tubes and set the PCR machine to do the first step (the reverse transcription) which takes about 45 minutes, and when that is finished, add the remaining reagents and set the machine for a labeling-amplification step (about 2 hours); at that point, you can walk away. The labeled probes can be stored at -20„aC or used straightaway.
In my opinion, the best qualities of this product are the reproducibility, ease of use and the high quality of the labeled probes which can be easily tested following the manufacturer¡¦s instructions. I personally go straight away to the hybridization, as the results are very good every time. Of course, it is important that the biotin used is of good quality, and if using the recommended one the kit will work perfectly.
If there is something I don¡¦t like, it is the ¡§just enough¡¨ amounts of reagents that are included in the kit; this does not allow any room for error. I agree that if used as per the instructions, it should be enough, but you do get the odd day when things don¡¦t go quite to plan and there is a need to repeat the labeling.