Amplify and Biotinylate RNA for Use on Illumina® Sentrix® Arrays
|Illumina® TotalPrep™ RNA Amplification Kit |
RNA amplification has become the standard method for preparing RNA samples for array analysis [1–2]. The Illumina RNA Amplification Kit was developed for use with Illumina Sentrix arrays. It is based on the RNA amplification protocol developed in the laboratory of Dr. James Eberwine . The procedure consists of reverse transcription with an oligo(dT) primer bearing a T7 promoter and in vitro transcription of the resulting cDNA with T7 RNA Polymerase to generate hundreds to thousands of antisense RNA copies of each mRNA in a sample. The Illumina RNA Amplification procedure maintains a close concordance to the basic Eberwine protocol. The labeled cRNA produced with the kit is ready for hybridization with Illumina arrays.
The new TotalPrep RNA Amplification Kit incorporates extensive improvements to the in vitro transcription reaction (IVT) over the previous Illumina RNA Amplification Kit. The IVT reaction has been optimized for the most effective biotin labeling, and an NTP mix containing biotinylated UTP has been added to the kit, allowing ease of use and optimal labeling. These modifications to the kit have significantly increased sensitivity, providing increased Percent Present calls and single round amplification with just 50 ng of input RNA. Figure 1 demonstrates the amplified RNA yields possible from inputs ranging from 10–100 ng from two different sources.
The kit contains reagents for 24 single-round amplification reactions.
- Kacharmina JE, Crino PB, Eberwine J (1999) Preparation of cDNA from single cells and subcellular regions. Methods Enzymol 303:3–18.
- Pabon C, Modrusan Z, Ruvolo MV, Coleman IM, Daniel S, Yue H, Arnold LJ Jr (2001) Optimized T7 amplification system for microarray analysis. Biotechniques 31(4):874–9.
- Van Gelder RN, von Zastrow ME, Yool A, Dement WC, Barchas JD, Eberwine JH (1990) Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc Natl Acad Sci USA 87: 1663–7.