Biologically relevant effects of mRNA amplification on gene expression profiles

Abstract

Background: Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix.

Results: Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other.Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P <0.001). Those reporters were also predicted to form significantly more (0.76+/-0.07 versus 0.38+/-0.1) and longer (9.4+/-0.3 versus 8.4+/-0.4) hairpins as compared to representative control reporters present before and after amplification.

Conclusion: This study establishes that the gene expression profile obtained after amplification of mRNA of left ventricular biopsies is representative for the whole left ventricle of the rat heart. However, specific gene transcripts present in parental tissues were undetectable in the minute left ventricular biopsies. Transcripts that were lost due to the amplification process were not randomly distributed, but had higher GC-content and hairpins in the sequence and were mainly found in the lower intensity range which includes many transcription factors from specific signalling pathways.

Figure 1

Two weeks after surgery, contraction rate (+dP/dt) (a) and relaxation rate (-dP/dt) (b) of 6 rat hearts from which biopsies were taken were compared to data obtained from 6 sham-operated rats. Data were obtained by direct LV pressure measurements using a micro-tip pressure transducer (Millar Instruments, Tx, USA) inserted into the left ventricle. Data were obtained at baseline as well as during dobutamine-stimulated conditions.

Figure 2

Representative Bioanalyser Picochip plot of RNA isolated from one biopsy, with a 28S/18S ratio of 1.43.

Figure 3

Quantitative real-time PCR (qPCR) of biopsy RNA before and after amplification using a high (cyclophilin A), medium (ribosomal protein S9, RPS9) and low (collagen VI alpha 3, COL6a3) abundance transcript. Material for the 'before amplification' qPCR was obtained from cDNA synthesis from total biopsy RNA; material for the 'after amplification' qPCR was obtained from cDNA synthesis from first round cRNA. After amplification, biopsy #3 yielded aberrant Ct values for all 3 genes; biopsy #5 only for COL6a3.

Figure 4

Gene array data analysis. (a) Summary of gene array characteristics, as described in the results section "Amplification results in non-random loss of gene detection". Low intensity means a signal of less than 3000 on the Affymetrix gene chip. (b) Signal intensity plot in which every gene is represented by a square. Signals of parental LVs #1 and #2 are similar, and gene expression data of LV biopsy #2 are visualized in the squares as 'not changed' or 'false negatives'. Biopsy reporters predominantly disappear in the lower expression region of their parental LVs, as exemplified by LV biopsy #2. Data on LV biopsy #4 and 6 are similar.

Figure 5

Reporters that were not detected after amplification ■ had a significantly higher mRNA GC-content (53.7% ± 4.0 vs. 47.8% ± 5.5, P <0.001) (a), and contained significantly more (b) and longer (c) hairpins as compared to representative control reporters present before and after amplification o (0.76 ± 0.07 vs. 0.38 ± 0.10 hairpins per gene resp., P < 0.01; and 9.4 base pairs (bp) ± 0.3 per hairpin vs. 8.4 bp ± 0.4 resp., P < 0.05. A hairpin must contain 7 or more base pairs. The compared reporters were taken from the same region of expression (same signal intensities).

Figure 6

Ratio between present calls (heart/biopsy) and absent calls (heart/biopsy) calculated for the different GC%. GC% was subdivided in groups of 10%. Grey bars indicate the average ( ± SEM) of the three comparisons between parental LVs and LV biopsies; black bars represent the ratio of detection calls when the two parental LVs or all three LV biopsies have the same detection call.

Figure 7

Visualization of low abundance reporters that disappeared after amplification in 1 to 3 LV biopsies (indicated with different shades of red), as compared to the expression profiles found in the parental LVs with the computer application GenMAPP. MAPPs with the highest z-scores in GenMAPP are shown; they represent four major signalling pathways.