Amplification of RNA for Microarrays.

Abstract

Gene expression profiling typically requires microgram quantities of mRNA, which can be difficult to obtain. In such cases, RNA must be amplified to have enough material for microarray labeling and hybridization. This protocol generates amplified antisense RNA (aRNA) from limited quantities of total RNA. It is designed around maximizing yield and product length while minimizing template-independent side reactions. Template-independent reactions compete with the desired template-dependent reaction, an undesirable situation that becomes more severe as less RNA template is used. Amplification products dominated by template-independent product result in greatly reduced sensitivity and compression of differences in microarray hybridization experiments. Most notably, the oligo(dT) primer used in reverse transcription (RT) yields a high-molecular-weight product in the in vitro transcription (IVT) reaction independent of any cDNA template. This protocol therefore limits the amount of primer used by using small cDNA synthesis volumes. In addition, high-molecular-weight, template-independent product is generated in the presence of biotinylated NTPs and the absence of any polymer when excessive amounts of T7 RNA polymerase activity are used. Template-dependent product of questionable molecular weight and limited functionality in downstream reactions can also be produced with excessive T7 RNA polymerase activity. Essentially, more yield is not always better. Here, a single round of amplification typically results in a fivefold to 20-fold mass conversion of starting material. If the first-round aRNA is used as a template for a second round of amplification, 200- to 400-fold amplification is typical.