Small Sample Preparation
The first sample preparation protocols for use with DNA microarrays required huge amounts of RNA for each array. Over the past 10 years we have seen a significant reduction in starting material requirements. These initial protocols typically relied on converting mRNA to dsDNA and then performing a linear amplification using T7 RNA polymerase in an “in vitro transcription” reaction or IVT. The efficiency of these protocols has steadily improved over the years to the point where users can comfortably (and reliably) use 250 ng or less RNA for starting material.

However, many users are able to obtain only much smaller quantities of RNA for their studies. In 2006, we validated and adopted protocols using a new linear amplification technology (called ribo-SPIA) which allowed us to start with 5 ng of RNA to make a single stranded cDNA product. More recently this technology has been updated to reduce the starting requirement further (500 pg) and by using random primers to allow whole transcript amplification which is very resistant to problems with RNA degradation. A cartoon of the ribo-SPIA process is shown below.