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DNA Array Core Facility

RNA Amplification

An increasing demand to analyze expression from enriched cell types acquired by technologies such as laser capture microdissection and cell sorting, or the analysis of RNA from biopsies, is rapidly making RNA amplification necessary. As with all nucleic acid manipulation, RNA amplification assays require optimization for the generation of product which accurately represents the original transcript population. We routinely use two in-vitro transcription (IVT) techniques in the Core Facility:

Standard Amplification Protocol:

The standard amplification protocol is best for amplifying at least 500ng of total RNA and typically results in greater than 30ug of cRNA depending on the source and amount of input RNA. In rare instances the standard amplification protocol has been shown to yield sufficient amounts of cRNA starting with only 300ng. However, if starting from less than 500ng of total RNA, a subsequent round of amplification may be required to generate an adequate amount of material. Multiple rounds of amplification result in the loss of the 5' end of the transcript.

Reduced Volume Amplification Protocol:

The reduced volume amplification protocol, developed by Baugh et al. (2001), specifically reduces the cDNA synthesis volume and is designed to minimize undesirable side reactions while maximizing yield and product length. One example of an undesirable side reaction is primer artifact reproducibly generated when using the standard protocol. By reducing the reaction volume, the Baugh protocol dramatically reduces the amount of primer used, thus minimizing the amount of artifact produced. To maximize 5' complexity, the T4gp32 single stranded binding protein is added to the reverse transcription reaction to maintain template linearity for maximum length cDNA synthesis.