ARTICLE |
Correspondence to: Karoly Gulya, Dept. of Zoology and Cell Biology, University of Szeged, 2 Egyetem St., POB 659, Szeged H-6722, Hungary. E-mail: gulyak@bio.u-szeged.hu
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Summary |
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In estimations of mRNA copy numbers, quantitative in situ hybridization (ISH) is expected to be performed at saturating probe concentration. In practice, however, this condition can rarely be fulfilled when medium to high amounts of transcripts exist and/or in large-scale experiments. To resolve this problem, we developed and tested a double-step procedure involving the use of calmodulin (CaM) I, II, and III [35S]-cRNA probes on adult rat brain sections; the hybridization signals were detected with a phosphorimager. By means of hybridization at increasing probe concentrations for a time sufficient for saturation, saturation curves were created for and maximal hybridization capacity (Hmax) values were assigned to selected brain areas. The Kd values of these various brain areas did not differ significantly, which allowed the creation and use of one calibration graph of Hmax vs hybridized [35S]-cRNA values for all brain areas for a given probe concentration. Large-scale ISH experiments involving a subsaturating probe concentration were performed to estimate Hmax values for multiple CaM mRNAs. A calibration graph corresponding to this probe concentration was created and Hmax values (expressed in ISH copy no/mm2 units) were calculated for several brain regions via the calibration. The value of the method was demonstrated by simultaneous quantification of the total accessible multiple CaM mRNA contents of several brain areas in a precise and economical way. (J Histochem Cytochem 48:893904, 2000)
Key Words: rat brain, gene expression, calmodulin mRNAs, quantitative in situ hybridization, phosphorimager, image analysis, Hmax
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Introduction |
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Quantification of biomolecules is essential for the revelation of cell processes involved in developmental, physiological, and pathological changes. Although the sensitivity of the conventional ex vivo quantitation methods has been improved substantially (
[35S]-UTPS-labeled riboprobes are preferentially used for quantitative ISH because of their advantageous features such as sensitivity, specificity, and single-cell resolution. When the aim is to quantify target mRNAs in absolute terms, target sequences are expected to be saturated with labeled nucleic acid probes. However, this condition can rarely be achieved with [35S]-cRNA probes when medium to high amounts of cell transcripts exist and/or in large-scale experiments, because of the practical problems associated with the synthesis of large molar quantities of riboprobes with high specific activity. Probes with high specific activity are required for quantitation of areas in a non-homogeneous tissue with relatively low amounts of the target mRNA. Employment of a mathematical approach to estimate the maximal hybridization signal attainable under given experimental conditions circumvents the use of large amounts of probes. For estimation of the maximal hybridization signal from a signal measured only when a subset of target sequences forms hybrids, maximal hybridization signal values should be calibrated to the measured labeling values. The problems associated with the creation of a calibration graph vary according to the medium containing the probes. When the target sequence is in solution, the accessibility of each target molecule is the same. Therefore, only one saturation curve has to be created. This curve can readily be calibrated because the maximal hybridization signal can be directly related to the absolute amount of target mRNA which, in turn, can be simply measured by independent methods (e.g., spectrophotometry, fluorimetry). For membrane blots, the same situation can be assumed for practical purposes. For single cells, it can be presumed that the accessibility of mRNAs among individual cells of similar type is the same. As regards the calculation method, an explicit theory has been put forward (
In the present study, labeling values were converted to molar quantities (expressed in [35S]-cRNA ISH copy no/mm2 units; for explanation of the ISH copy number, see the Discussion) for precise correction for the tissue background, and the maximal hybridization signals were converted to maximal hybridization capacities (Hmax; in ISH copy no/mm2 units). The Hmax of a tissue area was defined as the maximal molar quantity of a [35S]-cRNA probe (expressed in ISH copy numbers) hybridized in unit area (1 mm2) of the tissue under the experimental conditions employed.
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Materials and Methods |
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Experimental Animals and Tissue Preparation
All animal experiments were carried out in strict compliance with the European Communities Council Directive (86/609/EEC) regarding the care and use of laboratory animals for experimental procedures, and also followed the Hungarian legislation requirements. Male SpragueDawley rats (260 ± 10 g) maintained under standard housing conditions were decapitated and the brains were quickly removed, embedded in Cryomatrix embedding resin (Shandon Scientific; Pittsburgh, PA), and frozen immediately at -70C. Serial coronal cryostat sections (15 µm) from selected brain areas were cut (according to
cRNA Probes
Rat genomic DNA was isolated by standard procedures (S (1250 Ci/mmol; ICN Biomedicals, Costa Mesa, CA) was incorporated by using Riboprobe System-T7 and Riboprobe System-SP6 (Promega; Madison, WI) according to the manufacturer's instructions. Labeled probes were purified by size-exclusion chromatography on a ProbeQuant G-50 Sephadex microcolumn (Pharmacia Biotech; Uppsala, Sweden) and the specific activities of the complementary sequences of CaM I, II, and III antisense cRNA probes were determined to be 6.6442 x 107, 4.6095 x 107, and 3.1236 x 107 cpm/pmol (i.e., 9.3047 x 108, 6.1837 x 108, and 6.1561 x 108 cpm/µg), respectively. The specific activities of the synthetized CaM I, II, and III sense cRNA probes were 6.7063 x 107, 7.6421 x 107, and 5.6726 x 107 cpm/pmol, respectively.
Membrane Standard Scales
Membrane standard scales were prepared as described previously (
In Situ Hybridization
The protocol for hybridization with [35S]-cRNA probes was essentially the same as described previously (
Phosphorimaging and Image Analysis
Brain and tissue paste sections and membrane standards were apposed to an SR Cyclone storage phosphor screen (Packard Instruments; Meriden, CT) at RT for 22 hr. Images were captured at 600 x 600 dpi resolution with the Cyclone Storage Phosphor System (Packard) and analyzed by the computer program OptiQuant version 3.0 (Packard). Images of membrane scales, tissue paste standards, and brain areas were outlined on the computer screen and their signal intensities were expressed in digital light units (dlu) per mm2 and corrected for the screen background (less than 7000 dlu/mm2). The final results were expressed in net dlu/mm2 values.
Quantitation was performed through several consecutive steps. First, it was assumed that identical concentrations of radioactivity in brain sections and brain paste standards resulted in identical net dlu/mm2 values in their images (
where RES is the result (ISH copy number/mm2), LAB is the labeling intensity (net dlu/mm2), AVO is the Avogadro number (6.02252 x 1023 copy number/mol), LRF is the labeling vs radioactivity factor [(net dlu/mm2)/(cpm/mm2)], RDF is the radioactive decay factor, and SPA is the specific activity of the probe (cpm/pmol).
Correction for the tissue background was performed by taking into account the specific activity of the cRNA probes. The screen background-corrected labeling value of a brain region hybridized with the antisense probe was converted to a RES value and then corrected for the average of the RES values of the same brain region hybridized with a similar concentration of the corresponding sense probe. The resulting net RES value (ISH copy no/mm2) was regarded as an estimate of the copy no/mm2 of the mRNA to be quantified.
Data Processing
Saturation curves were created and Hmax values were calculated with the computer software GraFit 3.0 (
where y is the [35S]-cRNA probe hybridized (ligand bound; in ISH copy no/mm2 units), H is the capacity for hybridizing the cRNA probe [Hmax, in ISH copy no/mm2 units; analogous to the capacity for binding the ligand (Bmax)], Kd is the dissociation constant, and L is the concentration of the nonhybridized [35S]-cRNA probe (free ligand). In practice, saturation curves were created by plotting ISH copy numbers of hybridized cRNA in a 1-mm2 area of a selected brain region vs the concentration of the cRNA probe in the hybridization solution in fmol/ml units. The amount of probe bound in one section was determined to be less than 2.5% (at reasonable probe concentrations it was less than 1%) of the total amount of probe applied to the section (see Fig 3). Thus, saturation curves were created by using the total probe concentration instead of the free probe concentration.
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Data reduction and linear regression were accomplished with the computer software Excel 7.0 (Microsoft; Redmont, WA). Analysis of significance was carried out with the two-tailed Student's t-test. ISH copy number/mm2 values of the same brain area determined by saturation and calculation were considered significantly different when p was less than 0.05.
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Results |
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Linear Relationship Between Tissue Paste and Membrane Scales
Tissue paste and membrane standards were co-exposed to the storage phosphor screen, the resulting images were then quantified, and the signal intensity values were plotted against radioactivity, as shown in Fig 1. Regression analysis resulted in the equations yb = abx for the brain paste standard and ym = amx for the membrane standard, where x is the radioactivity (cpm/mm2), y is the signal intensity (net dlu/mm2), and ab (= 5872.51) and am (= 3612.17) are the slopes of the brain paste and membrane standard graphs, respectively. In consequence of the wide linear dynamic range offered by the storage phosphor screen technology (
Control Experiments for In Situ Hybridization
The specificity of the CaM I, II, and III cRNA probes was verified previously by Northern blotting analysis (
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Saturation of Brain Areas: Hmax
Determination of the Hmax values of brain areas by hybridizing a large number of sections with subsaturating concentrations of probe requires the establishment of Hmax values of selected brain regions by saturation hybridization experiments on a limited number of sections. Careful selection of the appropriate areas to be quantified in saturation experiments is a crucial issue. To minimize the amount of probe used for saturation, it is suggested that regions be chosen that display only minor alterations within a feasible range of the rostrocaudal axis of the brain, so as to facilitate the cutting of similar serial coronal sections. Selected areas must exhibit hybridized cRNA values covering a range as wide as possible (encompassing the hybridized cRNA values of all the brain areas to be quantified later by the calculation of Hmax) and distributed evenly within this range (to facilitate linear fitting).
Brain areas fulfilling these criteria were chosen and serial (approximately halving) dilutions of antisense and sense [35S]-cRNA probes for CaM I, II, and III mRNAs were hybridized with the sections (Fig 2). Because the amount of the probe bound to the section was found to be less than 2.5% (at reasonable probe concentrations it was even less than 1%) of the total amount of the probe applied to the section (Fig 3), the total probe concentration was used for the creation of saturation curves, instead of the free probe concentration. In the digital images of the sections, selected brain regions were outlined and measured and saturation curves were created for them by plotting tissue background-corrected hybridized cRNA values against probe concentration (Fig 4A4C). The saturation curves were then used to determine the Hmax values of the regions. Because multiple target CaM mRNA sequences were of medium to high abundance in the adult rat brain, high probe concentrations were needed for saturation. This often resulted in disproportionately heavy and patchy labeling of the sections hybridized with the highest concentration of probe (Fig 2), possibly in consequence of the physical sticking of the probe to the sections. Therefore, the determination of Hmax values only from measurements on saturated sections might well have led to biased results.
Transformation of Saturation Data
For the calculation of Hmax values of areas from hybridized cRNA values obtained from hybridization at a lower probe concentration, it was necessary to examine whether or not the Kd values of the saturation curves corresponding to different brain areas differed. None of the Kd values of the saturation areas was found to differ from the others at a significance level of 0.01 (two-tailed Student's t-test). At a significance level of 0.05, only one brain area, the lateral habenula (LHb), hybridized with the CaM I cRNA exhibited a Kd different from those of three brain regions (CA1, DG, and VPL+VPM; for abbreviations of brain areas see the legend to Fig 6), hybridized with the same riboprobe. Interestingly, in a repeated experiment, no Kd differed from the others at a significance level of 0.05. Therefore, we accepted that Kd was identical in the different brain areas, and defined Kav as the average of the Kd values for the brain areas hybridized with the same probe.
The constancy of Kd has important implications: with the use of Kav instead of Kd values and for a given L probe concentration, the equation used for saturation data fitting (see Materials and Methods)
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(1) |
can be rearranged for brain areas (1, 2, ..., n) in the form
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(2) |
Thus,
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(3) |
that is, the ratios of the hybridized cRNA values of different brain areas are identical with the ratios of the corresponding Hmax values at any L probe concentration. This justifies the estimation of Hmax from measured hybridized cRNA values.
This estimation can readily be performed by rearranging Equation 1 into the linear form
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(4) |
and multiplying the measured hybridized cRNA value (y) by the slope of the linear graph (Kav+L/L). However, use of this theoretically calculated slope of the Hmax vs hybridized cRNA graph gives less precise estimations (data not shown) than use of the slope of a similar graph based on experimentally determined Hmax and hybridized cRNA values. Such graphs corresponding to the probe concentrations used in saturation experiments can be created by plotting Hmax values vs hybridized cRNA values measured in saturation experiments (Fig 4D4F). The correlation coefficients of these experimentally determined calibration graphs remain high even at lower probe concentrations (Fig 5), allowing precise estimations of Hmax by measurements from a hybridization at a medium to low probe concentration.
Calculation of Hmax Using Data from Hybridizations at Subsaturating Probe Concentrations
To test the method of calculating Hmax, hybridizations were performed on rat brain coronal sections cut in the same plane as previously, but this time with a single, lower probe concentration (Fig 6). In the images of the sections, the same brain regions were quantified as above. Hmax values (determined previously in saturation experiments) were plotted vs the hybridized cRNA values, resulting in calibration lines (Fig 7A7C) corresponding to the probe concentration. Hmax for a brain region was then simply calculated by multiplying the hybridized cRNA value of the brain region by the slope of the calibration graph.
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The accuracy of the estimation method was checked by creating separate calibration graphs for each particular brain area quantified in the saturation experiments by omitting the data point corresponding to the brain area from the fitting of the calibration line. Calculated Hmax values provided good estimations for the corresponding Hmax values derived from saturation experiments (Fig 7D7F).
Hmax values for brain areas not quantified in saturation experiments and having hybridized cRNA values within the range quantified in the saturation experiments can be calculated in the same way as described above.
Summary of the Method
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Discussion |
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Recognition of the significance of alterations in gene expression requires the simultaneous localization and quantitation of nucleic acid sequences in tissues. The technique that is most promising as concerns acquisition of an answer to this commonly faced and technically demanding problem is quantitative ISH. To make a further improvement to the methodology of this powerful technique, we devised a simple and economical procedure for the simultaneous estimation of Hmax values for several areas in a heterogeneous tissue from hybridization data obtained at a subsaturating probe concentration.
Saturation of Areas in a Nonhomogeneous Tissue
By using the method presented above, we demonstrated that, under the experimental conditions we applied, the ratios of the hybridized cRNA values of different areas are constant, i.e., the accessibility of the mRNAs present in different regions of the tissue does not change as a function of the probe concentration (provided that the hybridization time is not a limiting factor). This allows use of the same calibration curve for each region in calculations of Hmax. Large differences in the permeability of different cell types, jeopardizing exact quantitation, are revealed by additional control experiments in which it is examined whether hybridization with a general probe, such as poly(U), labels all cells to a similar extent, or whether the ratios of the extents of labeling of the different regions under various fixation/proteinase digestion conditions are constant (
Hmax Calculation Method
In pharmacology, an exact determination of the binding capacity is achieved through the use of purpose-designed software rather than different forms of linear-ization of doseeffect curves (
For the estimation of predetermined Hmax values from measurements at a lower probe concentration, an arbitrarily chosen quasi-linear part of the saturation curve was accepted as a calibration line in previous studies (
Probe Copy Numbers and mRNA Abundance
Abundance of the target mRNA is estimated by hybridization methods, such as Northern blotting and ISH. However, use of these methods for the estimation of in vivo copy numbers necessitates several presumptions. (a) First, calculation of the specific activity of a [35S]-UTPS-labeled riboprobe requires the assumption that the RNA polymerase incorporates labeled and unlabeled nucleotides at the same rate (this is not valid for [35S]-UTP
S-labeled riboprobes [
This copy number is inevitably smaller than the in vivo copy number and does not correspond to any real subset of it. The in vivo target sequences fall into two categories: sequences not detectable at all by hybridization (lost during the steps of tissue removal, fixation, embedding and ISH, by washing out and/or degradation) and sequences having detectable and nondetectable segments [hybridization of the latter can be prevented by secondary nucleic acid structures, by steric hindrance, e.g., by crosslinks of fixatives, or simply by their degradation (
Despite the above-mentioned presumptions and the possibility of underestimation, there are significant advantages to the use of ISH copy number units. First, a precise correction for the tissue background can be made only by using molar quantities of probes. The performance of tissue background correction at the level of raw measurements (expressed in net dlu/mm2) or on the directly proportional data in cpm/mm2 units can result in negative background-corrected values for brain regions with low labeling when the specific activity of the sense probe is higher than that of the antisense probe. Then, the ISH-detected maximal copy numbers can be reproduced reliably by keeping the experimental conditions constant. This means that the ratios of the copy numbers detected by ISH, the copy numbers detected by Northern blotting, and the copy numbers of mRNAs present in vivo are constant, resulting in a constant ISHNorthern blotting calibration for a given probetarget mRNAISH protocol combination. The use of cpm or dpm values instead of ISH-detected maximal copy numbers would require a separate ISHNorthern blotting calibration for each ISH performed with a newly synthesized probe.
In this study, the ISH copy numbers refer to an area unit (1 mm2 surface area of a 15-µm-thick section). Similarly to the ISH copy number, this area unit is also relative: it can not be simply converted to a volume unit (mm3) by multiplying it by a volume factor (in this case by (1 mm3/0.15 mm2 = 66.6 mm), because change of the section thickness would modify the ISH copy number (due to alterations in target mRNA loss and probe penetration).
To reduce the difference between ISH copy numbers and in vivo copy numbers, we attempted to minimize the loss of the target sequences by utilizing quick embedding, cryosectioning, formaldehyde fixation (demonstrated to ensure the best compromise between target mRNA retention and accessibility;
Generalization of the Method
In this article we have presented and, using CaM I, II, and III cRNA probes, tested a method for the simultaneous determination of the attainable Hmax values of several regions in a heterogeneous tissue. Because the basic principles of quantitation extend over methodological boundaries, this technique can be of use not only in ISH experiments, with the application of a variety of probes, tissues, and detection systems, but in other histological methods also.
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Acknowledgments |
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Supported by grants from the National Scientific Research Fund, Hungary (OTKA T22822 to KG), and the National Council on Technical Development (OMFB 97-20-MU-0028 to KG).
We are grateful to Prof Laszlo Hatvani for his valuable comments on the manuscript. The skillful technical assistance of Ms Susan Ambrus is highly appreciated.
Received for publication July 26, 1999; accepted February 23, 2000.
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