Terminal Continuation (TC) RNA amplification enables expression profiling using minute RNA input obtained from mouse brain

Abstract

A novel methodology named terminal continuation (TC) RNA amplification has been developed to amplify RNA from minute amounts of starting material. Utility of the TC RNA amplification method is demonstrated with two new modifications including obviating the need for second strand synthesis, and purifying the amplification template using column filtration prior to in vitro transcription (IVT). Using four low concentrations of RNA extracted from mouse brain (1, 10, 25 and 50 ng), one round TC RNA amplification was compared to one round amplified antisense RNA (aRNA) in conjunction with column filtration and drop dialysis purification. The TC RNA amplification without second strand synthesis performed extremely well on custom-designed cDNA array platforms, and column filtration was found to provide higher positive detection of individual clones when hybridization signal intensity was subtracted from corresponding negative control hybridization signal levels. Results indicate that TC RNA amplification without second strand synthesis, in conjunction with column filtration, is an excellent method for RNA amplification from extremely small amounts of input RNA from mouse brain and postmortem human brain, and is compatible with microaspiration strategies and subsequent microarray analysis.

Keywords: TC RNA amplification; aRNA; brain extract; column filtration; in vitro transcription; microarray.

Figure 1.

Schematic representation of the aRNA (left panel) and TC RNA amplification (right panel) methods as they are applied to custom-designed cDNA array analysis.

Figure 2.

Comparison of the TC RNA amplification protocols and aRNA amplification using 50 ng of mouse brain RNA on specific gene expression levels. Key: APP, amyloid-beta precursor protein; CREB, cAMP response element binding protein; SOD1, superoxide dismutase; TUBB, beta tubulin.

Figure 3.

Custom-designed cDNA array analysis utilizing aRNA (A-D) and TC RNA amplification without second strand synthesis (E-H) in conjunction with drop dialysis purification.

Figure 4.

Comparison of the aRNA method (A-D) and the TC RNA amplification method (E-H) following column filtration purification using mouse brain RNA as an input tissue source.

Figure 5.

TC RNA amplification and aRNA amplification following drop dialysis (A, C) and column filtration (B, D) as a function of hybridization signal intensity (A, B) and percentage above negative control (NC) values (C, D). Single asterisk denotes (p < 0.05), double asterisk denotes (p < 0.01), and triple asterisk denotes (p < 0.001).