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Gene Expression Arrays:,,,Highly Sensitive Detection of Expression Patterns,,,with Improved Tools for Target Amplification


As the human genome project is completed, microarray technology offers the potential to study the genomes complexity. This technology facilitates the direct extraction of functional information from nucleic acids by measuring the RNA levels of a complete organism or its associated subsets.

After isolation of total RNA, various methods can be applied to prepare targets for microarray screening (Figure 1). The most common procedure involves direct cDNA labeling by reverse transcription in which fluorescencelabeled nucleotides are incorporated. A clear limitation of this technology is the large amount of RNA required per hybridization. Some of the most important applications in medical research involve studying very small amounts of tissues (e.g., microdissected tissue [LCM] or biopsy material from tumors). Therefore, target amplification methods have been developed to overcome this limitation:

    The linear cRNA amplification procedure, based on reverse transcription with an oligodT primer connected to a T7 promoter and in vitro transcription of resulting DNA with T7 RNA polymerase [1, 2], amplifies a RNA target approximately 100-fold. This method does not significantly distort the relative abundance of individual mRNA sequences within an RNA population [3, 4].
    A PCR amplification method based on reverse transcription, followed by random PCR amplification of the cDNA and in vitro transcription of the resulting PCR product with T7 RNA polymerase, is illustrated in Figure 2. With this procedure as little as 50 ng total RNA (from 1,000 cells or 0.1 mg of tissue) are required for expression profiling.

To investigate the bias of these amplification methods, we used yeast as a model eukaryotic organism due to its low level of splicing variants. The experiments included two different cell populations grown in rich and minimal media.

Material and Methods

Sample material
Saccharomyces cerevisiae was grown in YPD medium (50 g/l, rich) and SD base medium (26.7 g/l, minimal). Two percent D+glucose (carbon source) was added to both media. Cells were grown to an OD of 0.4 in a 1 : 4 dilution and collected from 40 ml of cell suspension. Total RNA isolation was performed as described in [5].

Target preparation /amplification

Direct cDNA labeling was performed according to the MWG PAN YEAST Array Application Guide. One hundred microgram of total RNA was labeled in a 40-l labeling reaction by using moloney murine leukemia virus (MMLV) reverse transcriptase and Cy3- and Cy5-dCTP (Amersham Biosciences). Linear amplification was performed with 10 g of starting total yeast RNA according to the package inserts of the cDNA Synthesis System, Microarray Target Purification Kit, and Microarray RNA Target Synthesis Kit (T7), using Cy3- or Cy5-UTP for labeling. Random PCR amplification was performed with 50 ng total yeast RNA according to the package inserts of the Microarray Target Amplification Kit, Microarray Target Purification Kit and Microarray RNA Target Synthesis Kit (T7), using Cy3 or Cy5 for labeling.

Hybridizati on
One microgram of Cy3- and Cy5-labeled cDNA or 10 g cRNA from the respective yeast population was used for hybridization. Hybridizations were performed overnight at 42 C using a buffer containing 50% formamide.

Yeast microarrays
Using a contact printing principle, 5-modified 40-bp oligonucleotides were covalently attached to the solid surface [6]. Each array was comprised of 6,250 oligonucleotides that detected different yeast open reading frames (ORFs).

Image analysis
Scanning for signal intensities was performed with a confocal laser scan microscope. Each slide was scanned six times per channel with an increasing photomultiplier setting to expand the dynamic range of the measurement. Resulting images were analyzed with the Imagene 4.0 Software; the output files were corrected between both channels via total signal intensities and local background subtraction using the MWG MAVI Software.

LightCycler RT-PCR
Quantitative RT-PCR was performed on the LightCycler Instrument using the DNA Hybridization Probe format. cDNA was produced from yeast total RNA in a 20-l reaction, including 20 g total RNA and a defined copy number of neomycin mRNA (spike-in control), oligo dT primer and avian myeloblastosis virus (AMV) reverse transcriptase. RNA and primers were first denaturated at 70 C for 10 minutes, then incubated at 42 C for 60 minutes, followed by a 5-minute denaturation at 94 C. Two hundred picograms and 20 pg of cDNA, primers and hybridization probes, and LightCycler FastStart DNA Master (as described in the protocol) were used in a 20-l reaction. The reaction conditions included: 10 minutes denaturation at 95 C, 45 cycles at 95 C for 10 seconds, 55 C for 15 seconds, and 72 C for 15 seconds. Fluorescence was monitored at the end of each 55 C incubation. The fluorescence detected in channel F2/F1 was analyzed using the LightCycler Analysis Software. The crossing point for each reaction was determined using the second derivative maximum algorithm and the arithmetic baseline adjustment. For quantification, a titration curve of neomycin mRNA was used.

Results and Discussion
Correlation of results obtained with and without amplification
Total RNA from Saccharomyces cerevisiae grown in rich or minimal medium was labeled with Cy3 and Cy5 by three different methods (Figure 1):
    100 g total RNA by reverse transcription (cDNA labeling/no amplification)
    10 g total RNA by linear amplification (cDNA synthesis followed by in vitro transcription)
    50 ng total RNA by random amplification (random PCR amplification followed by in vitro transcription).
Two hybridizations for each set of probes were performed on the MWG PAN YEAST Array, including a dye swap to minimize normalization and hybridization artifacts [7]. A threshold for low signal intensities was calculated based on the average signal intensities of 15 Arabidopsis-specific negative control spots. The average value of those 15 spots, plus the standard deviation multiplied by 1.96, gives a 95 % confidence that signals above this value are based on specific hybridization. Among a total of 6,250 spots, 75% (4,690) displayed signal intensities above the threshold. For those yeast ORFs, the average ratio of hybridization signals obtained from yeast grown in rich or minimal medium was determined. Within the 4,690 yeast ORFs, 562 ORFs displayed a ratio of ≥2, and 496 ORFs displayed a ratio o f ≤0.5 in the direct-labeling approach. These expression ratios were very reproducible for each target-labeling method applied. This is shown in Table 1 for the cDNA labeling procedure. The vast majority of the genes (97%) showed a less than twofold variation from the average expression ratio. Similar results can be obtained by comparing linear amplification with linear amplification, and random amplification with random amplification (data not shown).

The most important question is whether the three applied methods deliver similiar expression differences. Therefore we compared the cDNA labeling method with the amplification methods. Table 1 shows that 92% of the yeast genes show comparable expression alterations (≤ 2-fold variation) when cDNA labeling is compared to linear amplification. When cDNA labeling is compared with random PCR amplification, 89% of the yeast genes show similar expression rates. These results indicate that different amplification methods might not generate the same number of labeled target molecules from each template. Rather, the degree of amplification within a method is reproducible from one reaction to another. In addition, the different methods deliver similar expression alterations.

Validation of results with quantitative PCR on the LightCycler Instrument
To find out which target preparation methods would reflect the biological situation best, an independent method (quantitative RT-PCR) was applied to a number of genes. We randomly selected two genes from a group of upregulated genes and two genes from a group of downregulated genes in rich vs. minimal medium. We also selected three housekeeping genes that should show no alterations, and four genes from the group in which different methods show differences in the expression alterations. The group of u p- and down-regulated genes, as well as the houskeeping genes, show similar ratios (Figure 3). The yol 086c and yjl153c ORFs indicate that cDNA labeling and LightCycler quantification (to a higher degree) have a broader dynamic range.

For the genes varying in their expression levels, depending on the target preparation method used (Figure 3D), Light- Cycler analysis confirmed this result. Surprisingly, yor 091w seems to be down-regulated when using the cDNA labeling method, but is up-regulated when applying other methods. This was not expected since cDNA labeling is considered to introduce less bias. Nevertheless, this result was confirmed in an independent experiment (Figure 3E). Besides potential distortion of the relative abundance of individual mRNA by the method used, differences between amplified and non-amplified targets may also be due to different structures (RNA vs. DNA) and length. Targets produced by direct labeling of cDNA varied from 400bp to several kb in length. Amplified targets (cRNA) require fragmentation prior to hybridization.

Therefore those fragments vary from 50bp to several 100bp in length. Whereas 1 g of labeled cDNA could be applied on the array, 10 g cRNA is needed for sufficient signal intensities. Differences in target length, structure (RNA vs. DNA) and concentration can lead to differences based on the formation of secondary structures among different targets during hybridization as well as the variability in hybridization patterns.


The analysis of expression profiles from different organisms and cell types by microarrays is now being implemented in many laboratories. Based on the variable amount of starting material, we have shown that cDNA labeling, linear amplifica tion, and PCR-based amplification methods provide consistent and comparable results.



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