4016 Eric Gottwald1 Odilo Müller2 Andreas Polten2 1

Forschungszentrum Karlsruhe, Institut für Medizintechnik und Biopyhsik, Karlsruhe, Germany 2 Agilent Technologies Deutschland GmbH, Waldbronn, Germany

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Semiquantitative reverse transcription-polymerase chain reaction with the Agilent 2100 Bioanalyzer We have applied a method to monitor mRNA expression in a semiquantitative fashion on the Agilent 2100 Bioanalyzer. The method was originally described in 1994 by Wong et al. and referred to as the „primer-dropping“ method. This polymerase chain reaction (PCR) technique uses multiple sets of primer pairs in a coamplification reaction that amplifies the target of interest within a predetermined range specific for each target. Separation, detection and quantification of PCR products were accomplished using the Agilent 2100 Bioanalyzer in conjunction with the DNA 500 and the DNA 1000 LabChip kits for the detection of DNA fragments with a maximum size of 500 and 1000 bp, respectively. Using primers specific for the inducible form of hsp72 and primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal standard we were able to rapidly monitor and quantify inducible hsp72-mRNA expression. Keywords: Primer-dropping / LabChip / Bioanalyzer / Capillary electrophoresis / Heat shock protein 72 EL 4667

1 Introduction In April, 1983, Kary Mullis took a drive on a moonlit California mountain road and changed the course of molecular biology. During that drive, he conceived the polymerase chain reaction (PCR) [1]. Since then, not only the amplification and detection of the nucleic acid of interest but also the quantification is becoming increasingly interesting. During the following years, quantitative [2–6] as well as semiquantitative protocols [7–10] have been worked out. These techniques for DNA quantification worked well but problems have to be solved that are inherent to these methods. The quantitative methods which use external standards, depend on the availability and quality of these standards. In the case of semiquantitative conditions with internal standards, starting template abundance could be very different. This could lead to a plateau phase for the amplification of the first product, while the other is still in the exponential phase of the reaction. As a result, no reliable quantifications can be performed. The situation is even more complicated when standards are amplified in a separate reaction because one has to deal with considerable tube to tube or amplification efficiency variations, especially when reactions are carried out near saturation level, i.e., high

Correspondence: Dr. Eric Gottwald, Forschungszentrum Karlsruhe, Institut für Medizintechnik und Biophysik, Postfach 3640, D-76021 Karlsruhe, Germany E-mail: [email protected] Fax: +49–7247–82–5546 Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcription

ª WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001

cycle numbers. Because of the exponential nature of the reaction, very small differences in the amplification efficiency can result in profound changes on the abundance of the final product [11]. These principle problems were addressed by Wong et al. [11] in 1994, who performed an elegant set of experiments to monitor simultaneously multiple mRNAs within the same sample. The authors were able to show the serum-inducible expression kinetics of cell cycle-regulatory genes (cyclins A, B1, D1, and E) as well as immediate-early genes (c-fos, c-myc and b-actin). In this way, semiquantitative data could be obtained for each specific mRNA species. Since then, reverse transcription-(RT)PCR techniques have developed further and quantitative data can nowadays be obtained by real time PCR. This technique monitors the increase in the amount of amplified products directly. On the one hand, this can be achieved by using intercalating dyes which generate fluorescent signals upon binding to dsDNA in direct proportion to the amount of dsDNA [12]. On the other hand, hybridization probes can be used which make use of the fluorescence resonance energy transfer (FRET) effect [13]. These hybridization probes transfer energy to a nearby quenching molecule upon irradiation which prevents fluorescence. During amplification bound hybridization probes are cleaved by the 5’-exonuclease activity of the polymerase. The fluorescing and quenching dyes are separated making FRET impossible, thereby increasing the fluorescence signal in proportion to the rate of probe cleavage. Even probes for multiple DNA species in the same sample can be detected by appropriate choice of probes. 0173-0835/01/1810–4016 $17.50+.50/0

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Semiquantitative RT-PCR with the Agilent 2100 Bioanalyzer

However, since real time-PCR is expensive in instrumentation and consumables and the design of appropriate probes can be time-consuming, many researchers still use the classical quantitative RT-PCR approach via slabgel electrophoresis. In this approach, only the end-point concentration of expressed genes, which are amplified via RT-PCR, is measured. In order to get (semi-)quantitative results, a housekeeping gene is often coamplified with the gene of interest. Up- or downregulation of the gene of interest is monitored by comparing the relative intensities of the separated bands. If the expression of the investigated genes differs substantially, a so-called primer-dropping method can be employed [11]. Here, the number of PCR cycles can be adjusted for each gene individually, by adding primers at different points during the PCR reaction. In the present study, the use of the Agilent 2100 bioanalyzer for semiquantitative RT-PCR using the primer dropping method was investigated. Using lab-on-a-chip technology, the instrument allows for rapid separation of dsDNA and delivers higher quality data than classical slab-gel electrophoresis. Especially the quantification is greatly improved compared to gel due to the high sensitivity and the large linear dynamic range of the assay leading to improved results in the area of comparative RT-PCR measurements.

2 Materials and methods

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according to the manufacturer’s instructions. In brief: gel matrix was prepared by adding 25 mL of dye to a gel vial. The gel/dye mixture was filtered through a spin filter. The chip was filled with the gel/dye mixture and 9 mL of gel/ dye mixture were filled into the buffer wells. Sample and ladder wells were filled with 5 mL of DNA marker solution before adding 1 mL of ladder and sample in the respective wells. The chip was vortexed and placed in the bioanalyzer for analysis.

2.3 Cell culture We used the human Caucasian hepatocyte carcinoma cell line HepG2, ECACC Ref. No 85011430, for the experiments. The cells were maintained in minimum essential medium (MEM) (Invitrogen BV, Groningen, The Netherlands) supplemented with 1% nonessential amino acids solution, 2 mM L-glutamine, 100 units/mL penicillin, 100 mg/mL streptomycin (all from Invitrogen BV, Groningen, The Netherlands) and 10% fetal calf serum (PAA Laboratories GmbH, Linz, Austria). Cell expansion was performed using 250 mL tissue culture flasks (Falcon, Becton Dickinson Labware, Plymouth, England). Cells were passaged every 7 days at a density of 0.56106. For RT-PCR experiments 0.256106 cells were seeded onto 6 cm tissue culture plates (Falcon, Becton Dickinson Labware) and cultivated for three days. Tissue culture plates and flasks were maintained under standard culture conditions, i.e., 377C, 100% relative humidity, 5% CO2/95% air.

2.1 Chip electrophoresis

2.2 DNA assays For analysis of PCR products, the DNA 500 LabChip kit and the DNA 1000 LabChip kit (both Agilent Technologies, Waldbronn, Germany) were used. All chips were prepared

2.4 Heat shock For the determination of heat shock mRNA expression kinetics cells were cultured under heat shock conditions. The incubator was set to 427C with 100% relative humidity, 5% CO2/95% air and the cells were incubated for 30 min. After heat shock the cells were cultivated further at 377C for 0.5, 1, 2, 3, 4, 6, 8, 24, and 31 h whereafter total RNA was isolated as described.

2.5 Proprietary stimulus for hsp72 induction Apart from increased temperature, heat shock genes can be induced by a variety of stress stimuli, e.g., anoxia, heavy metals, ethanol, amino acid analogues, agents capable of perturbing protein structure as well as DNA tumor viruses. Heat shock proteins (HSPs) are thus called, more accurately, stress factors. We performed a series of experiments with a proprietary stimulus, different from elevated temperature, to monitor hsp72 expression under these conditions.

Miniaturization

All chip-based separations were performed using the Agilent 2100 bioanalyzer, which has been described elsewhere [14]. The bioanalyzer is a novel type of instrument that uses lab-on-a-chip technology to perform gel electrophoresis in the confines of a microfabricated chip. Electrical voltages are applied to fluid reservoirs by means of individual electrodes, each of which is connected to a separate high voltage power supply. Samples are separated electrophoretically in an entangled polymer solution similarly to capillary electrophoresis. The instrument allows for highly sensitive laser induced fluorescence detection using an intercalating dye, which is added to the polymer. The bioanalyzer software automatically calculates size and concentration of each separated band and displays the results in real-time.

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2.6 RNA preparation Cells plated onto 6 cm tissue culture plates were lysed with 400 mL lysis buffer (4 M guanidinium isothiocyanate, 25 mM Na3-citrate, 0.5% N-lauryl-sarkosine; from Sigma, St. Louis, MO, USA) containing 15 mL b-mercaptoethanol. The lysates were stored at –207C until use. For semiquantitative RT-PCR analysis total RNA of the lysates was isolated with spin columns (RNeasy Mini Kit, Qiagen, Heidelberg, Germany) according to the manufacturer’s protocol.

Electrophoresis 2001, 22, 4016–4022 number by the “primer-dropping” method [11]. PCR was performed on a conventional thermocycler and cycle conditions were as follows: 957C for 30 s, 557C for 30 s, 727C for 30 s. PCR was performed with the cycle numbers indicated. The reaction products were separated, detected and quantified with the Agilent 2100 bioanalyzer using the DNA 500 and DNA 1000 LabChip kit (Agilent Technologies Deutschland GmbH).

3 Results and discussion 2.7 Reverse transcription reactions RNA concentration was determined spectrophotometrically (GeneQuant, Amersham Pharmacia Biotech, Uppsala, Sweden). The samples were diluted to a concentration of 0.4 mg/mL RNA and 2.5 mg of total RNA was used for the reverse transcription reaction. The reverse transcription reaction was performed in 50 mL containing 2.5 mg of total RNA, 16GeneAmp PCR-buffer II with 2 mM MgCl2, 0.2 mM of each deoxynucleotide triphosphate (dNTP; Amersham Pharmacia Biotech AB), 20 U recombinant RNase inhibitor (Promega, Madison, WI, USA), 2.4 U M-MLV reverse transcriptase (Invitrogen BV) and 2.5 mM Oligo d(T)16 (Perkin Elmer, Norwalk, CT, USA) or primers specific for the gene of interest and incubated for 1 h at 397C followed by 5 min at 957C.

2.8 PCR reactions For PCR reactions primers specific for the inducible form of hsp72 were used. Primers were derived from the GenBank sequence M11717 and generated with the help of “Gene Fisher-Software” [15]. The primer pairs yield PCR products of 384 and 650 bp, respectively. Primer sequences for the 384 bp PCR product are (forward and reverse) : 5’- GTG CAG TCG GAC ATG AAG CA -3’, 5’CAG GAT GGA CAC GTC GAA GG -3’, primer sequences for the 650 bp PCR product are: 5’-GCA CTG GCC TTT CCA GGT GA-3’, 5’-TCG GAA CAG GTC GGA GCA CA3’. Because of its ubiquitous nature, its abundance and high degree of homology between species, we chose glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal standard with the following primer pair combination [16] which yields a 443 bp PCR product: 5’-TGA CCT TGC CCA CAG CCT TG-3’, 5’-CAT CAC CAT CTT CCA GGA GCG-3’. All primers were synthesized by Amersham Pharmacia Biotech AB. The PCRs of 25 mL contained 1 mL of RT reaction product, 16PCR buffer II with 2 mM MgCl2, 10 mM deoxynucleotide triphosphates, 2.5 U AmpliTaq polymerase (Perkin Elmer), 0.25 mL DMSO and 1 mM of the specific primer pair. Equal aliquots of secondary primer sets were added at the appropriate cycle

3.1 Amplification of GAPDH and hsp72 When using internal standards, such as GAPDH or hypoxanthine-guanine-phosphoribosyl-transferase (HPRT), the starting amount of these mRNAs usually exceed that of the gene of interest. This can be shown by amplification of these genes under identical cycling conditions in separate reactions. Figure 1 (lanes 1–3) shows a comparison of the amounts of GAPDH PCR product (443 bp) and hsp72 PCR products (384 bp and 650 bp) when both are amplified with 30 cycles independently. As can be seen, GAPDH shows the strongest signal intensity. When both cDNAs are coamplified this leads to a significant depression of the hsp72 PCR product bands (Fig. 1, lanes 4 and 5). Obviously, different amounts of template cDNAs result in competitive interference of PCR reactions which in turn leads to a loss of exponential amplification and therefore an attenuation of the final product levels [11]. The high abundance of GAPDH compared to hsp72 mRNA in nonstimulated cells requires either a reduction of starting material or reduction in amplification cycle numbers for GAPDH amplification.

3.2 Titration of the optimal cycle numbers for GAPDH and hsp72: the primer-dropping method Reduction of starting material by specific priming during the RT-reaction and then diluting the cDNAs for amplification is laborious and time consuming. A more pragmatic approach is to determine the cycling conditions for each gene of interest at subsaturing levels to assure exponential amplification. This was done in a titration curve for which GAPDH and hsp72 were coamplified with the cycle numbers indicated (Fig. 2). Cycling conditions were chosen such that hsp72 was amplified for 25 cycles whereas GAPDH was amplified with cycle numbers varying from 24 to 13 cycles. The Agilent 2100 bioanalyzer not only separates the two PCR fragments, it also performs automated sizing and quantitation of each band. The amount

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Figure 1. Gel-like image showing the results of separate amplifications and coamplifications of GAPDH and hsp72 in unstimulated HepG2 cells. Primers for GAPDH yield a PCR product of 443 bp (lane 1), primers for hsp72 yield PCR products of 384 and 650 bp (lane 2 and 3), respectively. Lane 4 and 5 show the results of the coamplification reactions. Due to the increased amount of GAPDH starting material, almost no peak could be detected in lane 4 (inset) and no peak could be detected in lane 5. Lane 6, N (negative control). Cycling conditions were 30 s 957C, 30 s 557C and 30 s 727C for 30 cycles.

Table 1. Quantitative analysis of the data that are shown in the gel image of Fig. 2 Cycle numbers

HSP 72 (nmol/L)

GAPDH (nmol/L)

25/24 25/23 25/22 25/21 25/20 25/19 25/18

9.24 9.00 16.13 10.21 16.87 14.89 11.36

7.50 3.24 3.59 0.70 0.75 0.40 0.00

The mean hsp72 concentration during the coamplification reaction can be determined to 12.5 6 1.17 nmol/L. The maximum value can be determined to 16.9 nmol/L and the minimum value to 9.0 nmol/L.

of PCR product is calculated in ng/mL as well as molarity (nmol). The results from the titration study are shown in Table 1. As can be seen the hsp72 PCR product is constantly amplified over a range of at least 7 cycles,

whereas the amount of GAPDH PCR product is increasing exponentially (Fig. 3). Suitable cycling conditions were determined to be 25 cycles for hsp72 and 20 cycles for GAPDH.

3.3 Monitoring hsp72 expression after heat shock We next sought to apply the PCR conditions with the parameters determined earlier on cells that were heat shocked at 427C for 30 min to monitor hsp72 expression kinetics. The precise concentration measurements from the chip-based separation allowed to calculate relative amounts of the amplified genes. As shown in Fig. 4, hsp72 expression is increased as early as 30 min after the end of the stimulus. Hsp72 concentrations rise from 6.53 to 33.08 nmol/L (Table 2) which corresponds to a 6.6-fold increase when normalized to GAPDH levels. Increased levels are detectable up to 3 h whereas from 4 h to 31 h at 377C no increased hsp72 expression can be detected.

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Figure 2. Gel-like image generated from the electropherograms of the 12 CE runs displaying a coamplification of hsp72 (384 bp) and GAPDH (443 bp) for 25 cycles and 24–13 cycles, respectively. As can be seen, GAPDH could be amplified exponentially whereas hsp72-concentrations remained at a relatively constant level over at least 7 cycles.

Table 2. Quantitative data showing the increase in hsp72 mRNA levels after heat shock

Con 30 min 1h 2h 3h 4h 6h 8h 24 h 31 h

GAPDH (nmol/L)

hsp72-5/6 (nmol/L)

22.52 17.26 16.98 12.21 18.64 23.45 24.23 24.94 32.19 29.69

6.53 33.08 25.78 12.98 9.06 3.58 5.89 7.76 3.83 5.49

Table 3. Quantitative data showing the increase in hsp72 mRNA levels after proprietary stimulus

% 15.13 100.01 79.22 55.47 25.36 7.97 12.68 16.24 6.21 9.65

Hsp72 mRNA levels were normalized to GAPDH levels which results in relative hsp72 mRNA levels given in percent. Con, control; time points indicate RNA isolation after the displayed hours at 377C.

Con 30 min 1h 2h 3h 4h 6h 8h 24 h 31 h

GAPDH (nmol/L)

hsp72-5/6 (nmol/L)

22.89 23.02 19.95 20.20 22.38 19.40 14.06 19.06 18.17 16.80

4.34 4.89 3.68 4.84 7.42 9.98 7.27 12.91 7.33 16.88

% 18.87 21.14 18.36 23.85 33.00 51.20 51.46 67.41 40.15 100.00

Hsp72 mRNA levels were normalized to GAPDH levels which results in relative hsp72 mRNA levels given in percent. Con, control; time points indicate RNA isolation after the displayed hours at 377C.

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(650 bp PCR-product) is completely different from the one with heat shock. Hsp72 induction begins 3 h after the end of the stimulus and increases up to 31 h (Table 3).

4 Concluding remarks

Figure 3. Quantitative analysis of the coamplification reaction of Fig. 2. hsp72 could be amplified at a constant level whereas GAPDH was amplified exponentially.

3.4 Monitoring hsp72 expression after proprietary stimulus We performed a series of experiments with a stimulus different from heat to analyze whether hsp72 can be induced at all and if so, whether the expression kinetics differ from the one of the induction by heat. As can be seen from Fig. 5, the time course of hsp72 expression

We have applied a method to monitor mRNA expression in a semiquantitative fashion on the Agilent 2100 bioanalyzer. By applying the so-called primer-dropping method we were able to show that lab-on-a-chip systems can be used for the rapid detection and quantification of gene expression levels in multiplex PCR reactions. In an exemplary fashion we could show that the method is able to monitor hsp72 expression in HepG2 cells. Upon induction by heat shock, we could show that hsp72 expression kinetics can not only be monitored, but also be quantified. Increases in hsp72 levels of more than 6-fold could be determined rapidly. By comparing internal GAPDH levels with the hsp72 expression level, we could generate semiquantitative RT-PCR results, thereby preventing problems inherent to external standards, like,

Figure 4. Electropherograms and the resulting gel-like image showing the hsp72 induction (650 bp) by heat shock (427C, 30 min). Total RNA was isolated at the indicated timepoints after stimulus end and reverse transcribed. Cycling conditions were 25 cycles for hsp72 and 20 cycles for GAPDH. Con, control before heat shock; N, negative control.

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Figure 5. Semiquantitative data of an hsp72 (650 bp) induction experiment after proprietary stimulus. Cycling conditions were 25 cycles for hsp72 and 20 cycles for GAPDH. As can be seen the kinetics after proprietary stimulus is different from the kinetics after 30 min heat shock. Con, control before heat shock; N, negative control.

e.g., tube-to-tube variations in amplification efficiency and quality of external standards. Additionally, we monitored the hsp72 expression kinetics after a proprietary stimulus different from heat and could show that the kinetics differed markedly. This could probably be due to a different activation of the hsp72 signalling cascade. It is known that hsp72 activation is mediated by a transcriptional activator [17]. The proprietary stimulus could activate the hsp72 cascade by a sideway resulting in a significant delay in hsp72 mRNA expresssion as was seen in our study. We would like to thank Ev Krüger, Simone Weigel and Mechthild Herschbach for excellent technical assistance. Received July 19, 2001

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