Amplified RNA degradation in T7-amplification methods results in biased microarray hybridizations

Abstract

Background: The amplification of RNA with the T7-System is a widely used technique for obtaining increased amounts of RNA starting from limited material. The amplified RNA (aRNA) can subsequently be used for microarray hybridizations, warranting sufficient signal for image analysis. We describe here an amplification-time dependent degradation of aRNA in prolonged standard T7 amplification protocols, that results in lower average size aRNA and decreased yields.

Results: A time-dependent degradation of amplified RNA (aRNA) could be observed when using the classical "Eberwine" T7-Amplification method. When the amplification was conducted for more than 4 hours, the resulting aRNA showed a significantly smaller size distribution on gel electrophoresis and a concomitant reduction of aRNA yield. The degradation of aRNA could be correlated to the presence of the T7 RNA Polymerase in the amplification cocktail. The aRNA degradation resulted in a strong bias in microarray hybridizations with a high coefficient of variation and a significant reduction of signals of certain transcripts, that seem to be susceptible to this RNA degrading activity. The time-dependent degradation of these transcripts was verified by a real-time PCR approach.

Conclusions: It is important to perform amplifications not longer than 4 hours as there is a characteristic 'quality vs. yield' situation for longer amplification times. When conducting microarray hybridizations it is important not to compare results obtained with aRNA from different amplification times.

Figure 1

Incubation-time dependent degradation of aRNA. 5 μg of total RNA were amplified and labeled (either with Cy3-UTP or Cy5-UTP) with the T7-System for 2, 4, 6, 8 and 16 hours. The aRNA was separated on a denaturing agarose gel and photographed on a gel-imaging system. Fainter bands in the Cy5-labeled aRNA are due to quenching of Cy5-fluorescence by SybrGreen II and lower yield. M=Molecular weight marker.

Figure 2

aRNA yield after different incubation times. 5 μg of total RNA were amplified and labeled (either Cy3-UTP or Cy5-UTP) or non-labeled ('no Dye' control) with the T7-System for 2, 4, 6, 8 and 16 hours (n = 3). The aRNA was purified by column chromatography and quantified by UV-spectrophotometry.

Figure 3

Incubation of aRNA in the absence of T7 RNA Polymerase. Cy3- or Cy5-labeled aRNA that had been synthesized by the optimal '4 hour protocol' was incubated under amplification conditions (37°C, amplification buffer) in the absence of T7 RNA Polymerase for 2, 8 and 16 hours. The aRNA was separated on a denaturing agarose gel and photographed on a gel-imaging system. M=Molecular weight marker. The slightly weaker band in Cy5 (8 h) might be due to small loading differences.

Figure 4

Hybridization signals with aRNA from different amplification times. Scatterplot of signal intensities (5760 features) from the hybridization with 4 h and 16 h amplified aRNA. The signals from the 4 h time-point were plotted in order of decreasing intensity (black) with the correspoding genes from the 16 h timepoint (red). The two transcripts (DLP10 and HGPRT) described further are marked in circles.

Figure 5

Signal intensities from triplicate hybridizations with aRNA from 4 h and 16 h amplification. Shown is DLP10 (spot #11) which undergoes significant signal loss. All other signals are from neighbouring spots to eliminate DLP10 signal decrease due to some spatial property of the slides.

Figure 6

Signal intensities from triplicate hybridizations with aRNA from 4 h and 16 h amplification. Shown is HGPRT (spot #12) which undergoes significant signal loss. All other signals are from neighbouring spots to eliminate HGPRT signal decrease due to some spatial property of the slides.

Figure 7

Scatterplot of log-ratios from the hybridization signals of 4 and 16 hour amplified aRNA. Ratios are displayed as a log₂-based MA-Plot according to [14] and were transformed by locally weighted regression [22]. Spots in circles are examples of the two transcripts (DLP10 and HGPRT) which undergo strong time-dependent degradation.

Figure 8

Hybridization images from the microarray features directed against the two transcripts from Fig. 7and the 'unaffected' transcript S27a. Circles indicate the degradation of the two transcripts by decrease of the signal.

Figure 9

Real-time PCR (Lightcycler™) based evaluation of the incubation time-dependent degradation of the two transcripts from Fig. 7and S27a. aRNA from the different incubation time-points (2 h, 4 h, 8 h and 16 h) was converted to cDNA and equal amounts were subjected to a real-time PCR with gene-specific primers (n = 3). The presence of a single PCR product was confirmed by melting curve analysis (data not shown) and agarose gel electrophoresis (inset; n.T. no template control; M 100 bp DNA marker). Numbers in columns refer to x-fold decrease compared to 4 h, calculated by a method described elsewhere [23].