Systematic analysis of T7 RNA polymerase based in vitro linear RNA amplification for use in microarray experiments

Amplification factors

Comparability of data sets obtained from amplified and non-amplified RNA

In 49 hybridisation experiments (tables 2 and 3), we compared the generalised log ratios of 7,347 genes using amplified or total RNA for labelling (figure 1). When labelled samples derived from total (Cy-3) vs. total (Cy-5) RNA were hybridised on 10 different microarrays, the boxplots, indicating the variation of the generalised log ratios, were generally wider in comparison to those of a second set of 10 microarrays with samples derived from amplified (Cy-3) vs. amplified (Cy-5) RNA (figure 1A). This result holds for the cases where differential expression is not expected (figure 1A, Ta and Tb compared to Aa and Ab) due to the use of the same tissue source for RNA isolation or whether it is anticipated (figure 1A, Tc and Td compared to Ac and Ad) since different tissues were used. These findings indicate a considerably lower variability of generalised log ratios of aRNA vs. aRNA hybridisations compared to total vs. total RNA hybridisations.

In a second experiment (figure 1B), we hybridised total RNA against aRNA from the same tissue samples on 15 microarrays. The result (figure 1B, group A) shows that the degree of variation of generalised log ratios is even larger than in the case of the total vs. total RNA hybridisation (figure 1A). Although no differentially expressed gene is expected in these experiments, the high variation of the generalised log ratios indicates decreased sensitivity of the measurement if total RNA is compared to aRNA on the same array. Therefore, different types of RNA (amplified and total RNA) should not be compared within one experiment. In contrast, only small variations were observed for the generalised log ratios of the corresponding 14 microarray hybridisations with aRNA vs. aRNA (figure 1B, groups B and C). In summary, the variability of the generalised log ratios of the 49 microarray hybridisations decreased from (1) total vs. aRNA, (2) total vs. total RNA to (3) aRNA vs. aRNA.

Linearity of amplification reactions

Prior to performing large scale microarray gene expression studies using RNA amplification, it is important to address the question as to whether the enzymatic modification of the RNA introduces a systematic bias to the list of differentially expressed genes. In other words: Is the amplification "linear"? To approach this problem, we hybridised 20 microarrays with samples derived from either total vs. total RNA or aRNA vs. aRNA from the same sources (table 2). The arithmetic mean of the resulting values from all hybridisations was determined for each gene and each channel separately (figure 2, Cy-3 channel not shown). In the ideal situation (100% correlation, identity line), the overall correlation coefficient should be equal to 1. The scattering of values around the identity line reflects the influences of gene specific RNA amplification factors and noise. For both channels, the scattering of values is homogeneously distributed on both sides of the identity line, which indicates an overall linear amplification. The correlation coefficients between total and aRNA calculated from the experiments were 0.87 and 0.89 for the individual Cy-5 and Cy-3 channels, respectively. Thus, there is a clear correlation between signal intensities resulting from non-amplified RNA as compared to amplified RNA, suggesting that aRNA hybridisations correspond to those obtained with unmodified samples.

Reproducibility of RNA amplification

Several other groups [8, 10, 14, 15] have applied Pearson correlation coefficients between log ratios in order to show the reproducibility of the RNA amplifications. High correlation coefficients are 0.8 or greater. We performed pairwise calculations of correlation coefficients using six hybridisations with aRNA samples (table 2, correlation). In result, the coefficients varied from 0.95 to 0.99 indicating a very high correlation between the individual amplification reactions.

A statistically solid way to estimate the reproducibility of hybridisations is the F statistic in which the ratio of variances between the different amplification groups is compared to that within the amplification groups. When the between variation is almost the same as the within variation, the logarithmic F-value comes to lie around zero, indicating high reproducibility; when the variation between is larger or smaller than within hybridisations, the F-value becomes larger or smaller than zero, respectively. To estimate the ratios, we performed four amplification reactions from the same total RNA source. The labelled products were hybridised in triplicate against an amplified reference sample on 12 microarrays (table 2, reproducibility). These experiments were carried out on two different days (table 2; day 1, arrays 58–61; day 2, arrays 62–69). For each gene from these hybridisations, an F-value was calculated and plotted in a histogram (figure 3). The negative median log F-value (-0.66) indicated a higher variance within than between the amplification groups. When only hybridisations performed on day 2 were considered, a median log F of 0.20 was found. These results suggest that other sources of variation (e.g. day-to-day variation, labelling, chip-batches) are of greater influence on the total variation of microarray hybridisations rather than the RNA amplification itself.

Correspondence of gene expression data obtained with amplified and total RNA

For the biological interpretation of the data, the crucial question concerning the use of RNA amplification methods is: Which proportion of the differentially expressed genes identified by hybridisation of total vs. total RNA corresponds to those found by hybridisation of aRNA vs. aRNA? To examine this question, we used eight microarray hybridisations (table 2), four of which were performed using total RNA vs. total RNA from different tissues, the other four with aRNA vs. aRNA derived from these total RNA samples. The generalised log ratios of four hybridisations were averaged and plotted against each other. For the highest 2,000 out of 7,347 genes, the correlation coefficient was 0.85 (figure 4). When low intensity genes were included, the correlation dropped to 0.72 (not shown), owing to the lower signal to noise ratios for these genes. The distribution of generalised log ratios in the aRNA hybridisations appears to be compressed (slope of 0.54 for the regression line), indicating a decreased sensitivity of the aRNA results compared to total RNA hybridisation data. Within the group of 2,000 highest expressed genes we also looked in each case, total RNA and aRNA experiments, at the 500 highest differentially expressed genes. The minimum fold change was 2.6 for total RNA and 1.8 for amplified RNA experiments. Out of 344 common genes (69%) all showed the same direction of regulation.

Tissue samples and RNA isolation

Tissue samples: Intraperitoneal injection of the human breast cancer cell line mcf7 into severe combined immunodeficiency (SCID) mice resulted in the growth of a solid tumour mass [16–18] varying in diameter from 1 to 7 mm. Six to nine weeks post injection, mice were sacrificed by application of CO₂, and the tumour mass was immediately frozen in liquid nitrogen. Human placental tissue was frozen as soon as possible. Total cellular RNA was isolated with the Trizol-method (TriFast, peqlab, Erlangen, Germany), following the manufacturers instructions, after homogenisation with a Mikro-Dismembrator S (Braun Biotech, Melsungen, Germany). The quality of the RNA samples was checked by the Agilent 2100 bioanalyzer (Agilent Technologies, Waldbronn, Germany). Only high-quality RNA samples (ratio 28S/18S rRNA > 1.8) were selected for the experiments.

RNA amplification

The cDNA synthesis was performed using a cDNA Synthesis System Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturers instructions. The ds cDNA was purified by phenol/chloroform extraction [19] precipitated in 5 M NH4OAc [19] and resuspended in 8 μl RNase-free water.

In vitro transcription [11] was performed using the AmpliScribeTM T7 High Yield Transcription Kit (Epicentre Technologies, Madison, WI, USA) according to the manufacturers instructions with the exception of increasing the incubation time to 4 hours. The newly synthesized aRNA was extracted in phenol/chloroform/isoamylalcohol as described above. Unincorporated dNTPs were removed by chromatography with MobiSpin S-300 columns (MoBiTec GmbH, Göttingen, Germany). Ethanol precipitation was performed as described in [19] and the resulting pellet was resuspended in 20–50 μl RNase-free water.

Microarray spotting

Glass slides used for this study carried 7,347 breast specific cDNA clones selected from the Human UniGene 1 clone set (German Resource Centre for Genome Research, Berlin, Germany). The clones were amplified by PCR and spotted in a "replicate array" design on silane-prep™ slides (Sigma Diagnostics, St. Louis, USA) to produce 14,694 individual spots. The slides were rehydrated, and the DNA was denatured with boiling water treatment prior to washing with 0.2% SDS, MilliQ- H₂O, 95% and 100% Ethanol. After the washing procedure, the microarrays were dried with compressed air.

RNA labelling and hybridisation

2 μg amplified antisense RNA (aRNA) were mixed with 500 ng random hexamer primers, incubated at 70°C for 10 min and cooled on ice. Alternatively, 10 μg total RNA were mixed with 500 ng (dT)₁₇ primer, incubated at 70°C for 10 min and cooled on ice. The labelling reaction was performed in a 12.5 μl reaction volume using 2.5 μl 5x RT buffer (Invitrogen, Karlsruhe, Germany), 1.25 μl 0.1 M DTT (Invitrogen), 1 μl dNTP-mix (dGAT, 5 mM each), 0.5 μl dCTP (3 mM), 0.5 μl RNasin (40 U/μl), 0.5 μl Cy-3 or Cy-5 labelled dCTP (1 mM, Amersham Biosciences, Freiburg, Germany) and 1 μl Superscript II reverse transcriptase (100 U/μl, Invitrogen). The mixture was incubated for 1 h at 42°C, and the reaction was stopped by addition of 1.25 μl 50 mM EDTA (pH 8). The input RNA was removed by hydrolysis with 5 μl 1 M NaOH at 65°C for 10 min, followed by neutralisation with 1 μl 5 M acetic acid. Cy-3 and Cy-5 labelled probes were combined, precipitated with isopropanol, washed with 70% ethanol, air dried and resuspended in 30 μl hybridisation buffer. The hybridisation buffer consisted of 350 μl 2x DIG-EASY (Roche Diagnostics), 210 μl MilliQ-H₂O, 70 μl 50x Denhardt's solution [19] and 70 μl Cot1-DNA (10 ng/μl). The 30 μl probe was heat denatured at 65°C for 2 min and hybridised to cDNA glass microarrays in a hybridisation chamber (Corning Inc., Acton, USA) over night at 37°C. After hybridisation the microarrays were washed at 25°C in a water bath with 1x SSC containing 0.1% SDS once for 15 min, and twice for 10 min. This was followed by washing twice with 0.1x SSC containing 0.1% SDS for 10 min. Washing steps were completed in 70% and 95% ethanol before the microarrays were dried.

Image quantification

The hybridised arrays were scanned with the GenePix 4000 B microarray scanner (Axon Instruments Inc., Union City, CA, USA), and the scanned images were analysed using GenePix Pro 4.0 software (Axon Instruments). Spot intensities were obtained by subtracting the median brightness of a region around the spot from the median of the region within.

Data analysis

For each of the datasets α ... η (table 2), the spot intensities were calibrated and transformed by the VSN method [20]. Differences between the resulting values are referred to as "generalised log ratios". All 14.694 spots from each slide were analysed, without filtering or thresholding procedures.

The reproducibility of different amplification reactions was measured using the F statistic to compare the variance for each gene within and between groups of hybridisations using amplified samples. The F value is defined as the ratio of the between and within groups mean squares mSS _B and mSS _W , whereas the mean square is defined as the sum of squares divided by the degrees of freedom:

given

and ,

where y _ikl denotes the measured value of the l th repetition of the k th amplification reaction for RNA transcript i. K denotes the number of all amplification reactions and L _k the number of replicas in group k.