An alternative method to amplify RNA without loss of signal conservation for expression analysis with a proteinase DNA microarray in the ArrayTube® format
© Schüler et al; licensee BioMed Central Ltd. 2006
Received: 30 March 2006
Accepted: 12 June 2006
Published: 12 June 2006
Recent developments in DNA microarray technology led to a variety of open and closed devices and systems including high and low density microarrays for high-throughput screening applications as well as microarrays of lower density for specific diagnostic purposes. Beside predefined microarrays for specific applications manufacturers offer the production of custom-designed microarrays adapted to customers' wishes.
Array based assays demand complex procedures including several steps for sample preparation (RNA extraction, amplification and sample labelling), hybridization and detection, thus leading to a high variability between several approaches and resulting in the necessity of extensive standardization and normalization procedures.
In the present work a custom designed human proteinase DNA microarray of lower density in ArrayTube® format was established. This highly economic open platform only requires standard laboratory equipment and allows the study of the molecular regulation of cell behaviour by proteinases. We established a procedure for sample preparation and hybridization and verified the array based gene expression profile by quantitative real-time PCR (QRT-PCR). Moreover, we compared the results with the well established Affymetrix microarray. By application of standard labelling procedures with e.g. Klenow fragment exo-, single primer amplification (SPA) or In Vitro Transcription (IVT) we noticed a loss of signal conservation for some genes. To overcome this problem we developed a protocol in accordance with the SPA protocol, in which we included target specific primers designed individually for each spotted oligomer. Here we present a complete array based assay in which only the specific transcripts of interest are amplified in parallel and in a linear manner. The array represents a proof of principle which can be adapted to other species as well.
As the designed protocol for amplifying mRNA starts from as little as 100 ng total RNA, it presents an alternative method for detecting even low expressed genes by microarray experiments in a highly reproducible and sensitive manner. Preservation of signal integrity is demonstrated out by QRT-PCR measurements. The little amounts of total RNA necessary for the analyses make this method applicable for investigations with limited material as in clinical samples from, for example, organ or tumour biopsies. Those are arguments in favour of the high potential of our assay compared to established procedures for amplification within the field of diagnostic expression profiling. Nevertheless, the screening character of microarray data must be mentioned, and independent methods should verify the results.
Proteinases play an essential role in numerous biological processes such as cell growth and differentiation, embryonic development, wound healing, antigen presentation etc., but also for pathological events like inflammation, coronary heart failure or cancer [1, 2]. Above all, tumour invasion and metastatic spread of tumours require the activity of proteolytic enzymes . Some proteinases have been identified as prognostic markers for the overall survival of a patient, as well as an indicator for a metastasis-free outcome post therapy. For instance there is evidence for a positive correlation between the expression of Matrixmetalloproteinases 2 and 9 (Gelatinases A and B) and a poor prognosis in almost all kinds of cancer . Urokinase (uPA), the urokinase receptor and the uPA inhibitors PAI-1 and -2 are enhanced in several female tumours [5–7]. Also, the Cathepsins B, L, K and S may play an active role in cancer development  and inflammation diseases like rheumatoid arthritis . Because of the importance of proteolysis in vivo, it is of great interest to better understand how the balance between active proteolytic enzymes and their endogenous inhibitors is controlled. Furthermore, it is also important to learn at which level (expression, activation, compartimentalization) a disturbed proteolysis is caused. The introduction of synthetic inhibitors of proteinases as therapy concepts drew high expectations for controlling or limiting the degradative potential of proteinases in e.g. cancer or rheumatoid arthritis . However, several aspects of such treatments are still uncertain. Expression analysis using DNA microarray technology is one potent tool to study the involvement of proteases in various normal and pathological processes or to follow the expression during an inhibitor treatment. However, it must be urgently stressed that such analyses require confirmation by independent methods such as e.g in vivo activity measurements of enzymes or at least QRT- PCR measurements.
Recent developments in DNA microarray technology led to a variety of open and closed devices and systems including high and low density microarrays for high-throughput screening applications as well as specific diagnostic purposes . Beside predefined microarrays for specific applications manufacturers offer the production of custom designed microarrays adapted to customers' wishes [11–15].
Array technology demands a complex procedure including both the reproducible and robust array production and the many steps in sample preparation (e.g. RNA extraction, amplification and sample labelling), hybridization and detection which may frequently lead to a high variability of the results between several and even equal experiments. Therefore, standardization and normalization procedures are very important and yet one of the most time-consuming steps in the development of new array based assays.
While analyzing RNA of clinical samples the amount of material available often limits the examination. Direct sample labelling via reverse transcription requires 20–100 μg total RNA  making this simple protocol inapplicable for most routine diagnostic processes of clinical samples. Two main concepts offer solutions for this dilemma. One possibility is to increase the labelling efficiency (meaning a higher signal per molecule ratio). This has been achieved using techniques like e.g. tyramide signal amplification and/or amino-allyl labelling [17, 18]. Other protocols amplify the RNA necessary for labelling in a different manner. One basic protocol is the amplification of aRNA (antisense RNA) by IVT (In Vitro Transcription), the so called Eberwine protocol . A variety of alternatives to this costly and lengthy procedure have been developed: SPA (Single Primer Amplification) , SMART technology (Clontech), Ribo-SPIA™ RNA amplification (NuGEN) , amplification using terminal continuation (TC RNA amplification)  etc. Numerous researchers investigated the conservation of differential expression and reproducibility by comparison of different amplification techniques, standardization and normalization of array results [23–28], respectively.
The main challenge is to maintain the differences of expression levels between different RNA species during the labelling procedure in a reproducible manner. We work with custom designed microarrays of low density in the ArrayTube® format. This offers a cost effective platform and requires only standard laboratory equipment and an array tube reader (atr01). By application of standard labelling procedures (e.g. Klenow fragment exo-, single primer amplification or IVT) we noticed a loss of some gene signals. In order to overcome this problem we developed a modified SPA protocol. We included target specific primers designed individually for each spotted target oligomer. Here we present the results by amplifying, labelling and hybridizing specific transcripts of interest.
Results and discussion
Comparison of different labelling methods
Labelling in the course of reverse transcription of as much as 5 μg total RNA resulted in non detectable or only very poorly detectable hybridization signals (results not shown). This was expected and corresponds to descriptions in literature and to protocols recommending 25–100 μg total RNA for direct reverse transcription labelling reactions . Therefore, we prepared samples according to the alternative and modified protocols, respectively.
Another possibility to explain the results was that they were based by the labelling procedure itself. Therefore, we decided – in contrast to the overall amplification with SPA primer – to try a specific linear amplification as described in the methods section where instead of the SPA primer specific primers for every target were used. Additionally, we incorporated biotinylated dUTP in the linear amplification reaction using the primer mixture and thus, diminished the number of steps required for sample labelling compared to original SPA protocol.
Optimization of the labelling protocol with specific primers
We investigated the influence of amplification temperature, primer concentration, reaction volume and ds-cDNA concentration in order to further improve the labelling protocol in the course of linear amplification with specific oligonucleotides. As one example, Fig. 3 shows the signal intensity and hybridization pattern at different primer concentrations at an annealing temperature of 62°C. For further investigations we have chosen a primer concentration of 60 nM each (final). It was applied in all assay investigations to prevent cross reactions and thus, false positive signals. In addition, that primer concentration was the lowest one yielding detectable hybridization signals of all expected signals. The assay was well functioning for that little primer set. This was in accordance with descriptions of a similar procedure to amplify genomic DNA with 39 oligos in a linear manner.
Experiments with an increased number of specific primers
Consequently, the behavior of the system was investigated with a stepwise increasing number of specific primers. Because of the complex reaction mixture cross hybridization reactions may be expected which had to be ruled out.
Group 1: Matriptase, PAI-1 and 2 (the initial test mix)
Group 2: MMP1, 3, 7, 13 and GAPDH
Group 3: MMP2, 9, 16, β-actin
Group 4: Cathepsins K and S, TIMP3, TIMP4
The single primer groups were used in amplification reactions. The hybridization patterns were compared with the results of PCR to omit false positive signals. Hybridization signals were confirmed by positive results of the respective PCR measurements. False positive signals were not detected. In the following and final experiment all available specific primers were combined and used for an amplification reaction. We compared the hybridization pattern of the three cell lines human fibroblasts, DLD-1, 97TM1, respectively, already chosen for the first experiments (see above). We noticed a remarkably improved specificity of the hybridization pattern compared to the traditional labelling protocols. The results were in accordance to the results of qualitative PCR experiments – the number of hybridization signals essentially corresponded with the number of positive signals in qualitative PCR.
Validation of results with real time measurements
Array analyses are extremely dependent on a high reproducibility. Most problems to be solved by an array analysis are based on the direct comparison of a normal sample with samples in which different expression is expected. Therefore, competitive hybridization where two different samples are labelled with e.g. Cy3 and Cy5, respectively, and afterwards mixed and hybridized again one and the same array is the mostly used technique in array based expression profiling. In our experiences the modification of only one part of the assay – e.g. just the polymerase used for amplification – or of the protocol, results in non- or hardly comparable array results. Not only the kind of labelling but also the conditions of the labelling protocol are very decisive for comparable and successful array experiments.
Comparative hybridization experiments – Affymetrix whole genome chip Hu133 plus 2.0 versus tube array
To elucidate the quality of the new protocol we performed comparative hybridization experiments with our assay and with the well-established Affymetrix whole genome chip Hu133. However, only 44 of the 49 targets present on the tube array were also present on the Affymetrix chip. We have chosen a stimulated and a non-stimulated sample (the established colon carcinoma cell line Coga-1  with and without Interleukin-6) to detect differences in the expression profiles and to compare the results achieved using the two different analytic systems. We were aware of the difficulties to compare two absolutely different systems concerning both the probe labelling protocol and the detection method: affymetrix chip results in absolute fluorescence values; array tube system applies a non linear enzymatic precipitation reaction (and thus a signal enhancement) to detect hybridization signals. The consequence is a much higher dynamic range for the affymetrix chip than the array tube may achieve. Nevertheless, the gene expression profile should be the same applying the different array systems, subject to the condition of high quality of analyses.
With one exception, all expressed genes detected with the Affymetrix chip (cut off: fluorescence value of 300, detection p-value < 0.065) were also found with the array tube, only MMP-14 which showed very high fluorescence values on the Affymetrix chip was not detectable at the array tube. However, the quantitative RT-PCR measurement confirmed the result of the array tube (no expression of MMP-14).
Hybridization signal strength of five genes found to be expressed using the array tube but not using the Affymetrix chip, respectively, compared with the results of quantitative PCR experiments (non-stimulated Coga cells). Array tube: relative transmission values, normalized overall. Affymetrix chip: relative fluorescence value, normalized overall
Relative expression level of selected genes (fluorescence and transmission values, respectively) versus expression level determined by quantitative RT-PCR (non stimulated cell line)
619; 976; 1564,5
However, the main application of array analyses is the direct comparison of two samples as stimulated vs. non-stimulated cells or normal vs. tumor tissue etc. For this purpose, we found a good correlation of the expression profiles determined with the two microarray systems/probe preparation protocols, respectively, and the quantitative RT-PCR results.
We designed a protocol for amplifying mRNA in a linear mode from as little as 100 ng total RNA. The protocol offers a simple method for detecting even low expressed genes by microarray experiments in a reproducible and sensitive manner. The central point of the method is the linear ssDNA amplification step with concomitant labelling. Preservation of signal integrity and the little amounts of total RNA necessary for this labelling protocol make that method applicable for investigations with limited amounts of material like clinical samples from biopsies or tumours.
DNA microarray design and preparation
For the manufacturing of the arrays, 3'-aminomodified oligonucleotides were purchased from Metabion (Martinsried, Germany). Oligonucleotides were used at a final concentration of 10 μM in Spotting Buffer 1 (CLONDIAG chip technologies GmbH, Jena, Germany) and spotted using a Microgrid II spotting machine (Genomic Solutions Ltd., UK) following the procedure supplied by the manufacturer. Every probe was spotted redundantly two times on the array. After production, arrays were inserted into ArrayTube™ reaction vials.
Probe sequences were derived from published sequences using the Array Design software package by Clondiag Chip Technologies (Jena, Germany). In the additional files 1, 2, 3 tables show target genes, sources of the sequence data, as well as probe sequences and array layout. Consensus regions in the alignments of all available sequences of each target were chosen for the probe design. The resulting sequences were selected to be specific for the target and to have similar length, GC content and melting temperatures in order to yield comparable signal intensities. The final probe sequences were again blasted against the database  to exclude false-positive reactions due to possible cross-reactivities or false-negative reactions due to sequence variations.
The site directed oligonucleotide set for the linear amplification procedure consisted of 94 oligonucleotides. It was designed according to the initial alignments for the probe design as described above. For each target, a consensus region was identified which was situated 5 to 50 bp upstream of the probe binding site (see additional files). Sequences with similar physicochemical parameters were chosen from these regions and used for primer design. The final primer sequences were blasted against the database  avoiding possible cross-reactivities as well as sequence variations. Primer sequences are also listed in additional files (additional file 4). Oligonucleotides were purchased from Invitrogen (UK). They were used as a stock solution mixture with concentrations of 1 μM for every individual primer.
The development of the Affymetrix microarray was performed in an independent routine lab according to the manufacturers recommendations. 5 μg purified RNA was used as starting material for this purpose.
Total RNA from cells and tissues was isolated with TRIZOL® Reagent (Invitrogen, UK) according to the suppliers' instruction. RNA was re-dissolved in RNase-free water. The quality of the isolated RNA was controlled using non denaturing 1.25% agarose gel electrophoresis and the determination of the A260/A280 ratio (GeneQuant II, Pharmacia Biotech). Only samples exhibiting no RNA degradation and showing an A260/A280 ratio equal or above 1.8 were used for further applications. Preparations were stored at -80°C.
Labelling by reverse transcription of total RNA
For direct labelling of cDNA via reverse transcription 10 μg of total RNA was mixed with 1.5 μl oligo-dT15-primer (0.5 μg/μl, Promega, Mannheim, Germany) and sterile water (final volume 14, 6 μl), denatured for 15 minutes at 65°C, then chilled on ice for 5 minutes. After a short centrifugation step a mastermix consisting of 6 μl 5× First Strand Buffer (Invitrogen), 2 μl 0.1 M DTT (Invitrogen), 0.7 μl RNaseOUT (Invitrogen, 40 U/μl), 2.5 μl 5 mM dNTPs (dATP, dCTP, dGTP, Fermentas), 1 μl 3.25 mM dTTP, 1.4 μl 1 mM Biotin-16-dUTP (ROCHE, Penzberg, Germany) and 1.8 μl MMLV-RT (200 U/μl, Invitrogen) was added to get a final volume of 30 μl. The reaction mixture was incubated for 2 h at 37°C, and afterwards stopped by adding of 1 μl 200 mM EDTA (final concentration ca. 7 mM). The sample was precipitated with 0.1 vol 4 M LiCl/2.5–3 vol ice cold absolute ethanol, washed with ice cold 70% ethanol and resuspended in 10 mM EDTA.
Labelling with Klenow fragment
After First Strand cDNA synthesis with oligo-dT15-primer (Promega) and MMLV-RT (Invitrogen) the resulting cDNA was precipitated with LiCl/ice cold absolute ethanol, washed with ice cold 70% ethanol and dried. The pellet was resuspended in 68 μl deionisized water. This mixture served as template for a labelling reaction with Klenow-Fragment, exo-. We used the BioLabel DNA-Labelling Kit (Fermentas) and followed the protocol provided by the manufacturer to generate a biotin labeled hybridization sample. This DNA was purified by precipitation (LiCl/Ethanol) for further applications.
The First Strand cDNA was prepared with an oligo-dT T7-primer (5'-AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC TTT TTT TTT TTT TTT TTT TTT TTT-3') and Superscript II RT (200 U/μl, Invitrogen) in a thermal cycler (10 minutes at 20°C, 60°C minutes at 37°C, final volume 10 μl). To generate the second strand an ice cold mastermix of 52.75 μl water, 7.5 μl 10×Second Strand buffer, 1.5 μl 10 mM dNTPs, 1 μl E. coli DNA-Ligase (5 U/μl, Fermentas), 2 μl DNA polymerase I (10 U/μl, Fermentas) and 0.25 μl RNase H (4.5 U/μl, Fermentas) was added to the cooled first strand tubes (final volume 75 μl). The double stranded cDNA (ds-cDNA) was purified by extraction with one volume phenol (once) and one volume of a chloroform/isoamyl alcohol mixture (24:1, once) and precipitated with 8 M NH4-acetate/ethanol at room temperature, washed twice with ice cold 70% ethanol and dried. The pellet was resuspended in 4 μl water. For In Vitro Transcription we used the Ambion Megascript kit (Ambion, USA). All components except the enzyme were allowed to come to room temperature. 1 μl of each component was added to the 4 μl ds-cDNA preparation and incubated for 16 h at 37°C. The resulting antisense RNA was purified with the RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol.
Single primer amplification (SPA) and labelling with Klenow fragment exo- was performed according to the original protocol published in .
Labelling in the course of linear amplification reactions
Double stranded cDNA generated according to the Eberwine protocol described above served as template for labelling during the linear amplification reactions. For overall amplification we used a primer with the sequence of the T7-promoter (called SPA primer, analogue to the SPA-protocol).
For specific amplification of selected targets we used amounts of ds-cDNA-preparations representing original amounts of 50–100 ng total RNA. An ice cold mixture of water, 1.5 μl MgCl2 (50 mM), 5 μl 10×PCR buffer (Invitrogen), 1.5 μl 5 mM dNTPs (dATP, dCTP, dGTP), 0.5 μl 3.25 mM dTTP, 0.86 μl 1 mM Biotin-16-dUTP (ROCHE), 0.6 μl Taq polymerase (5 U/μl, Invitrogen) and 3 μl primer mix (1 μM each) resulting in a final primer concentration of 60 nM each was added to the template (final volume 50 μl). The amplification was performed in a thermal cycler according to the following protocol: 3 min 94°C, 40 cycles: 1 min 94°C, 1 min 62°C, 2 min 72°C, cooling to 4°C. The reaction mixture was divided to get material for duplicates and used for hybridization without further purification steps.
Hybridization and detection
The array tube was conditioned by washing three times with 500 μl distilled water and once with 500 μl hybridization buffer 1 (Clondiag, Jena) at 25°C for 5 minutes. All steps were carried out using a horizontal shaker with temperature regulation (550 rpm, Thermomixer compact, Eppendorf, Germany). Biotinylated spike controls (0.05 μl each, equivalent to 6.7 × 10-6-1 × 10-5 nmol) were added to the samples as external control for hybridization, conjugation of enzyme and signal development due to TMB precipitation.
The samples were denatured at 95°C for 10 min and briefly centrifugated at 13000 rpm to collect the sample. 200 μl of pre-warmed hybridization buffer (50°C) was added to each sample and the resulting mixture transferred into the array tube. Hybridization was allowed to proceed on the shaker for 3 h at 50°C and 550 rpm.
The array tube was washed with 500 μl 2×SSC/0.01% Triton (5 min, 30°C), 2×SSC (5 min, 20°C) and 0.2×SSC (5 min, 20°C). After blocking with 100 μl of a blocking solution (2% milk powder, freshly prepared in 6×SSPE/0.005% Triton, 15 min, 30°C) the array was incubated for 15 min at 30°C with 100 μl of a 1:10 000 dilution of HRP Streptavidin (1 mg/ml, Clondiag, Jena) freshly prepared in 6×SSPE/0.005% Triton. Following that the array was washed twice with 500 μl 2×SSC/0.01 Triton (2 min, 30°C), twice with 500 μl 2×SSC and once with 0.2×SSC (5 min, 20°C).
The peroxidase pecipitation reaction (100 μl peroxidase substrate, Clondiag, Jena) was monitored by the ATR01 array tube reader (Clondiag, Jena) at 25°C recording 60 images (one image per 10 sec). Data analysis was carried out using IconoClust software Version 2.2 (Clondiag) determining the signal intensity and the local background value of each spot. The local background absorbance was subtracted from the absorbance of the spots. Only the average values of redundant spot hybridization signals with amounts above 0.05 (mean – local background values) were considered as positive. Both spotted oligos of an examined gene had to be "positive" to be considered as expressed. If these conditions were met, the signal resulting from the hybridization with the oligo sequence situated closer at the 3'end of the RNA sequence was used for further calculations.
Real time PCR (QRT-PCR) experiments
QRT- PCR experiments were carried out using a MyiQ™ Single colour QRT- PCR detection system (BIORAD, Herculas, CA). Reaction mixtures contained: 1 μl cDNA, 9.5 μl water, 2 μl primer mix (sense and antisense, 10 μM each), and 12.5 μl iQ SYBR Green Supermix (BIORAD, final volume 25 μl). A dilution series (10-2 - 10-6ng) of the specific PCR product of interest was prepared to determine the standard curve (absolute quantification). Template free controls served as a test of primer quality (formation of dimers etc.). First of all the melting curve of each target was measured to determine the optimal temperature for real time analysis (e.g. ß-actin: 72°C, PAI-1: 83°C, matriptase: 89°C, TIMP3: 85°C). The samples were amplified according to the following protocol: 3 min 95°C, 35 cycles: 20 sec 95°C, 40 sec 58°C, 1 min 72°C, real time data registration for 8 sec at the specific temperature determined before for each target. All samples were measured in duplicates and the right formation of the products was verified by agarose gel electrophoresis (1% agarose, unknowns, standards and no template control, product sizes: matriptase 470 bp, PAI-1 687 bp, TIMP3 445 bp). Real time data analysis was carried out using the optical system software version 1.0 supplied with the MyiQ™ real time instrument (BIORAD).
Primary human fibroblasts were isolated from arthritic patients according to the rules of the ethic commission of the Friedrich Schiller University Jena. Cells were cultured in DMEM/high glucose, 10% FCS, gentamycine 0.5 ml/100 ml (in triplicate, 75 cm2 flasks) until 80% confluence was reached. The medium was removed; the cells were washed twice with FCS free medium and further cultured in FCS free medium overnight. The medium was then removed, and 4 ml of fresh FCS free medium was used for stimulation experiments. The first sample served as control (FCS free medium without any cytokine. The second sample was stimulated with 20 ng IL6/ml medium, the third sample was stimulated with 2 ng TGFß/ml medium. Stimulation was performed for 24 hours. Medium was removed, cells were harvested and RNA was isolated with TRIZOL® reagent as described above. RNA quality was checked by non denaturing agarose gel electrophoresis and used for preparation of double stranded cDNA according to the first steps of the Eberwine protocol (see above). Additionally, an established human fibroblast line was also used for expression analysis.
Dulbeccos Minimum Essential Medium
double stranded cDNA
Fetal Calf Serum
Horse Radish Peroxidase
In Vitro Transcription
Moloney Murine Leukemia Virus Reverse Transcriptase
Polymerase Chain Reaction
Quantitative Real Time PCR
rounds per minute
Single Primer Amplification
Sodium Saline Citrate
Sodium Saline Phosphate Ethylenediaminetetraacetic acid
- TMB 3:
3', 5, 5'-Tetramethylbenzidin
This work was supported by grants of IZKF, Klinikum, Friedrich-Schiller-Universität Jena (Reg. Nr. B 307-04004) and Thüringer Kultusministerium (Reg. Nr. A 309-04001).
We thank R. Kinne (Institute of Experimental Rheumatology, University Jena) for primary human fibroblasts used for stimulation experiments, Swetlana Tsareva for RNA of different human cell lines and tissues and Waltraud Seul, Elke Müller, Ines Leube and Jana Sachtschal for technical assistance. The development of the Affymetrix micorarray was performed in the Institute of Vascular Medicine of our Klinikum (Prof. Habenicht) by M. Hildner whose assistance is thankfully acknowledged.
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