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Nucleic Acids Research, 2000, Vol. 28, No. 11 E54-e54
© 2000 Oxford University Press

Expression profiling across many samples via manifold-assisted mRNA processing

Anette Hagberg1, Gisela Barbany3, Henrik Krook2, Martina Samiotaki4 and Ulf Landegren1,3,*

1Department of Genetics and Pathology and 2Department of Clinical Immunology, Rudbeck Laboratory, Se-75185 Uppsala, Sweden, 3Department of Medical Sciences, Uppsala University Hospital, Se-75185 Uppsala, Sweden and 4Hellenic Anticancer Institute, Papanicolaou Research Center of Oncology and Experimental Surgery, 115 22 Athens, Greece

Received February 10, 2000; Revised and Accepted April 11, 2000.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 
Analysis of mRNA provides a condensed view of gene structure, and quantitative analyses can reveal induction of physiological or pathological gene expression programs. One of the main hurdles for routine mRNA analyses is the need to prepare large sets of samples in a rapid and standardized manner. We describe here a procedure for mRNA isolation and cDNA synthesis using manifold devices, consisting of a set of prongs that project into individual reaction wells. The prongs have a high binding capacity for the polyA-tails of mRNA and the captured mRNA is directly used to synthesize cDNA on the supports, followed by amplification. The convenience and reproducibility of the procedure allows profiling of gene expression over time, by comparing many different samples. Using the device mRNA was simultaneously isolated and accurately measured from up to 96 different samples of anywhere between 10 and 200 000 cells. The amounts of a leukemia-specific transcript could be measured when the malignant cells represented <=0.01% of the sampled cells. We illustrate the possibility of analyzing a number of tissues and monitoring expression of sets of cytokines, involved in rejection, at variable times after transplantation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 
Qualitative and quantitative investigations of expressed genes offer a view of the genome in action. Such procedures can demonstrate differences in expression levels between normal tissues and ones affected by disease. Isolated mRNA molecules also offer a concise overview of the structure of individual genes, for example mutation scanning. In many instances the expression of single or a few genes is of interest, and excellent means are now available to measure copy numbers of transcripts in mRNA samples, for example through reverse transcriptase–PCR (RT–PCR) with real-time detection via the 5' nuclease assay (1,2), or using molecular beacons (3). In other cases expression of large sets of genes are studied by sequencing parts of cDNAs (4), of even shorter snippets thereof in the SAGE procedure (5) or through hybridization of samples to arrays of immobilized oligonucleotides (6), or of cDNAs (7).

An important limiting factor for the routine application of expression analyses of individual or of sets of genes relates to the need to prepare mRNA from large numbers of samples. Several methods are in use for mRNA purification, such as organic extraction or trapping on solid supports coated with oligo(dT) oligonucleotides. Examples of available oligo(dT) supports include cellulose fibers (8), membranes, pipette tips (9) or particles, paramagnetic (10) or otherwise, but these methods have limitations as to how many samples can be handled at the same time.

We now present a means to standardize isolation and to accurately compare expression levels of mRNA from large numbers of samples, using sets of multipronged supports that fit into conical microtiter wells, and that have been coated with large amounts of oligo(dT)-cellulose in a simple procedure. These manifold devices permit isolation of mRNA from large sets of samples over a wide range of cell numbers and derived from different tissues, followed by cDNA synthesis and quantitative analysis of gene expression with minimal handling (Fig. 1). A similar strategy to bind biotinylated molecules to manifold supports has been previously described (11,12). The procedure allows comparisons of expression levels within and between different samples, and risks of degradation, contamination and sample mix-up are minimized. mRNA molecules <=14 kb in size were isolated and the amounts of transcripts could be monitored over a wide concentration range.



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  		Figure 1. A set of oligo(dT)-coated manifold supports for mRNA isolation placed in a 96-well microtiter plate.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 
Manifold supports
The manifold supports are polystyrene devices with eight conical tips shaped to fit 96-well microtiter plates used for amplification (ThermowellTM, Costar, NY) (Fig. 1). The supports were kindly prepared for us by Amersham Pharmacia Biotech (Uppsala, Sweden). A non-disposable holder allows 12 sets of supports to be manipulated in parallel, in order to process a total of 96 individual samples.

Coating of the manifold supports with oligo(dT)-cellulose
The surface of the manifold prongs was modified with cellulose fibers to which T<25 oligo(dT) had been conjugated. The plastic devices were sonicated in 95% ethanol for 20 min in a Metason Sonicator (Struer). Four grams of oligo(dT)-cellulose (type 77F, Amersham Pharmacia Biotech), was repeatedly rinsed in 4–5 vol triethylamine. The oligo(dT)-cellulose was suspended in a 70% (v/v) slurry consisting of oligo(dT)-cellulose in triethylamine. The tips of the sonicated solid supports were momentarily dipped in the stirred slurry and dried in air. Remaining unbound cellulose fibers were removed by washes with ethanol, and the supports were rinsed in distilled water and stored dry at 4°C until use. The oligo(dT)-coated supports have been stored for 6 months without any reduction in binding capacity. A similar procedure for conjugating large amounts of streptavidin molecules to plastic supports has been previously reported (12).

Cell lines and tissue samples
The K562 cell line was originally derived from a patient with chronic myelogenous leukemia (CML) (13). BSM is a lymphoblastoid cell line. Both cell lines were grown in RPMI 1640 containing 2 mM L-glutamine, 50 IU/ml penicillin, 60 µg/ml streptomycin, 100 µM HEPES pH 5.4 and 10% fetal calf serum. A primary culture of fibroblasts was established from a skin biopsy obtained from a normal individual.

Plasmid construct and in vitro transcription
A 362-bp fragment of the bcr–abl gene was cloned into plasmid pTML65(A30) (14). RNA molecules with A30-tails were generated by in vitro transcription of the NsiI-linearized plasmid in 40 mM Tris–HCl pH 7.9, 6 mM MgCl2, 2 mM spermidine, 10 mM NaCl, 10 mM dithiothreitol (DTT), 1.5 U/µl human placental ribonuclease inhibitor (HPRI), 0.5 mM rATP, rGTP, rCTP, 1 U/µl T3 RNA polymerase (Promega, WI) and 2.5 µCi/µl [{alpha}-32P]UTP. The reaction was incubated at 37°C for 60 min, followed by an incubation with 0.5 U/µl RQ DNase I (Promega) for 30 min.

Analysis of the binding capacity of manifold supports
The binding capacity of batches of manifold supports was determined by incubating individual prongs with a constant amount of the polyadenylated radiolabeled in vitro transcript, in the presence of increasing amounts of unlabeled A20 oligonucleotides in a total volume of 50 µl extraction buffer A (100 mM Tris–HCl pH 8.0, 0.5 M LiCl, 10 mM EDTA, 5 mM DTT, 1% lithium dodecyl sulphate). Unless otherwise stated buffer A was used in all the experiments.

After 30 min at room temperature the supports were washed eight times for 2 min by immersing in 70 µl of wash buffer (10 mM Tris–HCl pH 7.5, 1 mM EDTA, 0.1 M NaCl) in microtiter wells. The amount of in vitro transcript bound to the solid support was measured by scintillation counting (LS 3801, Beckman Coultier, CA).

Isolation of polyadenylated mRNA
Before RNA preparation, cells were washed in phosphate-buffered saline and suspended at a final concentration of 107 cells/ml in extraction buffer A. As an alternative, cells were lysed in extraction buffer B containing 10 mM Tris–HCl pH 7.9, 150 mM NaCl, 1.5 mM MgCl2, 0.65% NP-40 and 0.6 U/µl of HPRI.

Tissue samples (~5 x 2 x 2 mm) were collected at surgery and frozen at –70°C. The samples were homogenized in extraction buffer by mincing with a pair of scissors, followed by repeated passage through a 0.4 x 40 mm needle, after which the cell lysates were stored at –70°C.

Approximately 100 000 cell equivalents were aliquoted into individual wells of a microtiter plate and diluted in extraction buffer to a final volume of 50 µl. Oligo(dT)-coated supports, prewashed in extraction buffer A or B, were incubated in the wells for 30 min at room temperature, followed by eight washes of 2 min each in 70 µl wash buffer.

Instead of using oligo(dT), specific biotinylated primers can be bound to streptavidin-coated supports (12) and used to isolate the desired mRNA species. In general we have found this to be less successful, even under conditions where rare transcripts are searched for in large numbers of cells. This could reflect a greater accessibility of the polyA tails for hybridization to the support-bound oligonucleotides.

cDNA synthesis and amplification
After immobilization of the mRNA, the manifolds were transferred to 50 µl cDNA synthesis reactions containing 50 mM Tris–HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM dNTP, 0.25 µg/µl bovine serum albumin (BSA), 1 U/µl MMLV reverse transcriptase (Amersham Pharmacia Biotech), 0.6 U/µl HPRI (Amersham Pharmacia Biotech), and 1 µM random hexamers. The reactions were incubated at 37°C for 60 min, and the newly-synthesized cDNA was released from the supports by incubating the manifolds for 5 min in 70 µl of water at 95°C. The incubation was performed in a Peltier Thermal Cycler PTC-200 (MJ Research, MA). Random hexamers or specific antisense oligonucleotides were preferred over oligo(dT) for priming cDNA synthesis, in order for the cDNA to be released from the support by denaturation, and to ensure that the relevant part of the transcript was being copied.

Ten microliters of the cDNA was used in subsequent PCRs. The 50-µl amplification reactions contained 50 mM KCl, 10 mM Tris–HCl pH 8.3, 1.5 mM MgCl2, 0.0125 µg/µl BSA, 0.2 mM dNTP, 0.5 µM of forward and reverse primers and 1.25 U of AmpliTaq GoldTM (PE Biosystems, Foster City). The polymerase was activated by a pre-incubation at 94°C for 10 min, followed by 94°C for 30 s, 55°C for 30 s, 72°C for 45 s for 35 cycles and then 72°C for 7 min, performed in a Peltier Thermal Cycler PTC-200 (MJ Research). For quantitative measurements of the synthesized cDNA, a 5' nuclease assay was performed in the ABI PRISM" 7700 Sequence Detection System (ABI, Foster City). The results were represented as threshold cycle values, that is the estimated amplification cycle number when fluorescence exceeded a threshold value (2). For these reactions the polymerase was activated by a pre-incubation at 94°C for 10 min, followed by 94°C for 15 s and 58°C for 60 s for 50 cycles. For nested amplification, after the first 30 cycles a 50-fold dilution of the reaction was amplified another 40 cycles using a nested set of primers.

The sequences of amplification primers and of the probe for the 5' nuclease assays are shown in Table 1. Primers were designed to span exon–exon borders in order to avoid generating amplification products from genomic DNA.



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  		Table 1. Oligonucleotides used for amplification, and as probes in the 5' nuclease assays

R, primers intended for detection of rat cytokines.

 
mRNA expression analysis of cytokines after transplantation
Grafts of fetal porcine islet-like cell clusters were implanted under the kidney capsule in Lewis rats. Animals were sacrificed and grafts collected at 3, 5 and 12 days after transplantation. Grafts from individual rats were homogenized and mRNA was isolated, followed by cDNA synthesis and amplification with real-time detection using the 5' nuclease assay.

The ratio of transcripts for each of the cytokines IL2, IL4 and IL10 over those for ß actin were recorded for all samples (Table 1). The amounts of transcripts of cytokine and ß actin were estimated by comparison with standard curves of 5' nuclease threshold cycle values for known amounts of dilutions of all PCR products.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSION
 REFERENCES
 
Binding capacity of the supports
The capacity of individual prongs of the support for binding polyadenylated nucleic acids was measured by allowing increasing amounts of a 20mer oligonucleotide, A20, to compete with a radiolabeled in vitro transcript for binding to the supports. As illustrated in Figure 2, the addition of 40 pmol of unlabeled oligonucleotide decreased the binding of the labeled transcript by approximately one-half. This binding capacity was routinely obtained in different batches of the supports with half maximal binding at 48 ± 4 pmol SEM. The measure may not reflect the capacity of the supports for binding longer mRNA molecules isolated from tissue samples.



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  		Figure 2. Binding capacity of the manifold supports. A constant amount of radiolabeled in vitro transcripts was allowed to bind to the supports in the presence of variable amounts of a non-labeled competitor (dA) oligonucleotide. Each point represents RNA isolation on a separate prong.

 
The amount of mRNA that can be immobilized onto the support was estimated by comparing the binding capacity of the manifold support with the capacity of oligo(dT)25-coated dynabeads, (Dynal a.s., Olso, Norway). mRNA was isolated from 200 000 cells using the solid supports and a dilution series of Dynabeads. After reverse transcription the cDNA was amplified in the TaqMan instrument. RNA isolated on the manifold supports resulted in the same threshold value as 20 µl Dynabeads, corresponding to capacity 0.2 µg mRNA. Samples greater than 500 000 cells resulted in a proportionately less mRNA binding, perhaps reflecting the increased viscosity of the sample or that the binding capacity of the manifold supports was exceeded (data not shown). Using paramagnetic particles the sample preparation can more easily be scaled, compared to manifold supports, but manifold supports are easier to handle in routine analyses.

Kinetics of binding to the supports
A radiolabeled in vitro transcript of 386 nt was used to investigate the time required to bind mRNA to the supports. The manifold supports were incubated with the transcripts for variable times. In a parallel experiment we incubated the supports with K562 cell lysates. This cell line carries the translocated Philadelphia chromosome characteristic of CML. As a consequence of the translocation the two genes bcr and abl have become fused (15). The fused gene gives rise to a unique chimeric transcript, which can be used to monitor the disease at the mRNA level (16). We monitored the number of amplification cycles required to detect the transcripts of the bcr–abl fusion gene through real-time detection using the 5' nuclease assay.

A 60-min incubation was adequate to bind maximal amounts of the radiolabeled in vitro transcripts of the bcr–abl gene. This was also true for binding of the same transcript from the K562 cell line, as measured by the 5' nuclease assay (Fig. 3). The results of this assay were reproducible among individual mRNA isolations, followed by separate DNA syntheses and amplifications.



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  		Figure 3. Kinetics of mRNA-binding to the oligo(dT)-coated manifold supports. Lysates corresponding to 100 000 K562 cells were incubated with individual prongs, and bcr–abl transcripts (triangles) were reverse transcribed and amplified with real-time detection by the 5' nuclease assay by monitoring fluorescence from degraded 5' nuclease during amplification. The cycle numbers when fluorescence exceeded a threshold value were plotted as functions of time of incubation of the lysates with supports. A radiolabeled in vitro transcript (circles) was also incubated with individual prongs and bound transcripts were measured by scintillation counting. mRNA isolations from K562 cells were done in quadruplicate.

 
Isolation of transcripts from a variety of tissues
In order to establish the versatility of the mRNA isolation procedure, seven different transcripts were investigated in various tissues, including whole blood, in vitro cell lines, and solid tissue samples obtained at surgery. The isolated transcripts were amplified by RT–PCR and analyzed via agarose gel electrophoresis (Fig. 4). In particular, one of the longest mRNAs known, the 14-kb dystrophin (DMD) transcript previously shown to be expressed in the lung (17), was successfully isolated from lung tissue by binding to the oligo(dT) supports, reverse transcribed and detected by amplifying a sequence at the remote 5' end of the transcript. DMD transcripts were also detected in brain samples obtained at autopsy (results not shown). This demonstrates that very large mRNA molecules also can be isolated using the supports.



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  		Figure 4. Analysis of seven transcripts, analyzed via the manifold supports from six different tissues. The products of RT–PCR were analyzed by agarose gel electrophoresis. Controls without cDNA or with genomic DNA failed to give rise to amplification products (results not shown). M, molecular weight markers.

 
Isolation of mRNA from variable numbers of cells
It is helpful if widely different amounts of tissue samples can be accommodated in the same sample preparation procedure. In order to investigate this point we prepared lysates from the K562 cell line to isolate and estimate the amounts of transcripts derived from the bcr–abl fusion gene, in samples representing from 10 to 200 000 cells. The cells were lysed in either extraction buffer A or B.

Samples lysed in extraction buffer A became viscous as the ionic detergent ruptures the nuclear membrane, releasing DNA. To reduce viscosity the lysates were passed 10 times through a syringe fitted with a 0.4 x 40 mm needle. As an alternative, cells were lysed in extraction buffer B. The non-ionic detergent in this buffer preserves the nuclei intact, thereby the viscosity does not increase and nuclear transcripts are not released. The quantitative estimates were similar in the two buffers and accurately reflected the numbers of copies of the transcripts present in samples of 10–200 000 cells (Fig. 5).



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  		Figure 5. Isolation of mRNA from different numbers of cells. Variable cell numbers were lysed in the two different extraction buffers A (ionic detergent) and B (non-ionic detergent), and incubated with individual prongs. mRNA was isolated in triplicate, reverse transcribed and amplification products of the bcr–abl gene were measured in the 5' nuclease assay.

 
Measurement of transcripts present in a minority of cells
Valuable information about the functional state of a tissue sample can be obtained by estimating the expression levels of specific transcripts. A case in point is the bcr–abl transcript which is present in blood and bone marrow cells of individuals with CML. After clinical remission, residual transcripts can nonetheless frequently be detected using sensitive amplification-based detection. These transcripts reflect so-called minimal residual disease, and quantitative monitoring of the transcripts can alert the physician to an impending clinical relapse that necessitates prompt reinstitution of therapy (18). In a model experiment, K562 cells expressing the fusion transcripts were diluted among BSM cells that do not express this particular transcript. mRNA was isolated from a total of 100 000 BSM cells aliquoted in individual wells, followed by RT–PCR. Figure 6 demonstrates that the bcr–abl transcript could be measured using the 5' nuclease assay over a range extending down to 1 cell in 10 000 having the characteristic transcript among a total of 100 000 cells (Fig. 6). The observed linear relation between the logarithm of the proportion of leukemic cells and the amplification cycle in which the transcript was detected by the 5' nuclease assay demonstrates that the capacity of the supports is sufficient to measure the amount of the transcript even in the presence of a large amount of total mRNA.



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  		Figure 6. Measurement of bcr–abl transcripts from cells representing a variable proportion of the total number of cells added. K562 cells were diluted among BSM cells that do not express the bcr–abl transcript. mRNA was isolated, reverse transcribed, amplified and detected through the 5' nuclease assay.

 
Expression analyses of xenograft rejection in Lewis rats
Xenotransplantation could circumvent the problem of limited availability of suitable human donors for transplantation of islets producing insulin. As a baseline for a study with the aim of limiting transplant rejection we followed the transcription of three cytokines in rat recipients of pancreatic grafts. mRNA was isolated from transplants collected at various time points after grafting, and the levels of IL2, IL4 and IL10 were monitored. The highest expression levels of the cytokines were reached after 5–12 days (Fig. 7). IL10 was the most highly expressed gene, and IL2 increased sooner after transplantation compared to IL4 and IL10 mRNA. This supports an earlier study by Kovarik et al. (19).



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  		Figure 7. Expression analyses of three different cytokines involved in the immune response after xenotransplantation. The grafts were collected at 3, 5 and 12 days after transplantation. mRNA was isolated, reverse transcribed, amplified and detected through the 5' nuclease assay. The amount of cytokine expressed in a sample is presented as a ratio between transcripts of each cytokine and of ß actin. Values of three replicates mRNA isolations are connected by vertical bars.

 

   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSION
REFERENCES
 
Manifold supports coated with oligo(dT) can be easily manufactured and greatly simplify processing sets of tissue samples for gene expression analysis. Specific transcripts can be accurately analyzed qualitatively and quantitatively in samples representing a wide range of cell numbers. Excellent sample tracking is possible, allowing large numbers of individual samples to be handled in parallel with minimal risk of sample mix-up or contamination. cDNA synthesis of 96 samples can be completed within 2 h of sample collection, and the eluted cDNA can be analyzed directly or preserved as a permanent record of genes expressed in the sampled tissues. By now our supports have enabled many colleagues, as well as hundreds of students with little or no prior experience of molecular genetic work, to successfully isolate and analyze transcripts from patient samples. We have used the supports to perform thousands of individual mRNA isolations in projects to study patient responses to drug treatment, to monitor minimal residual disease in leukemia and to follow the course of immune responses during transplant rejection.

The supports could easily be adapted to handle larger numbers of samples still, or to be used in automated formats, and a similar procedure could be used for isolation of genomic DNA via suitable affinity matrices. Simplified sample preparation procedures as presented herein are a prerequisite for routine analyses of gene expression.


   ACKNOWLEDGEMENTS
 
Dr Lena Scheibenpflug kindly provided biopsy specimens, and Olov Korsgren provided transplants. We thank Maritha Mendel-Hartvig, Dr Anders Isaksson and Mats Gullberg for thoughtful comments on the manuscript. This work was supported by grants from the Beijer Foundation, the Swedish Medical and Technological Research Councils and the Swedish Cancer Foundation.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Department of Genetics and Pathology, Rudbeck Laboratory, Se-75185 Uppsala, Sweden. Tel: +46 18 471 4910; Fax: +46 18 471 4808; Email: ulf.landegren@genpat.uu.se Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 

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G. Barbany, A. Hagberg, U. Olsson-Stromberg, B. Simonsson, A.-C. Syvanen, and U. Landegren
Manifold-assisted Reverse Transcription-PCR with Real-Time Detection for Measurement of the BCR-ABL Fusion Transcript in Chronic Myeloid Leukemia Patients
Clin. Chem., July 1, 2000; 46(7): 913 - 920.
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