Polymerase chain reaction for detection of cytokine gene expression Anne O’Garra DNAX A powerful

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in Immunology

Introduction Cytokines are soluble mediators which play important roles in the regulation of growth and differentiation of leukocytes. We have obtained vast knowledge of cy tokine interactions using cell lines, but a more valid picture is obtained from the study of cytokine gene expression by cells isolated directly from the host during inIlammatoty and/or immune responses. However; there are a number of technical obstacles. Cytokine genes are generally expressed at low levels, their mRNAs have a short half-life [l] and the number of cells that can be obtained from primary in vivo sources is limited. The yield is drastically diminished if the cell populations are punfied further in order to identify the source of a particular cytokine (and/or group of cytokines). The acquisition of such information, however, is vital to the understanding of the role of cytokines in regulating immunity, and the development of cells that give rise to immune and/or inflammatory responses. The development of a method for cell-free DNA replication capable of producing selective amplification of a specific DNA sequence by a factor of lo6 [2], has enabled us to obtain such information. The polymerase chain reaction (PCR) uses two oligonu cleotide primers that flank the DNA sequence to be amplified. Repeated cycles of thermal denaturation of the DNA, annealing of primers to their complementary sequences, and primer extension give an exponential accumulation of the target fragments. The use of a thermostable DNA polymerase from 7i3ermu.s aquaticus (Taq) substantially improved the specificity, yield, sensitivity and length of targets that could be amplified, as well as simplifying the method and making it amenable to automation [2]. This PCR method has facilitated a variety of subsequent analytical manipulations on the resulting DNA, which are outside the focus of this review ([2];

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1992, 4:211-215

see also Steinman, this issue pp 205-210). The application of this technique to amplify cDNA prepared by reverse transcription of whole RNA mixtures [3] has allowed the analysis of cytokine mRNA expression from small numbers of cells. .A microadaptation of the guanidine thiocyanate/cesium chloride (GUSCN/CSCI) gradient ultracentrifugation technique [4,5] and the inclusion of carrier RNA has been of great help in the isolation of RNA from very small numbers of cells [ 5,601. Alternatively, RNA from cells which have been lysed with appropriate buffers may be reversetranscribed and specifically amplified using PCR. In this case, it is essential to use appropriate primers in the PCRreaction, in order to avoid the detection of genomic DNA (discussed below). In this review, we will discuss the various methods which have been described recently for qualitative and quanti tative detection of cytokine RNA expression in various models of immune regulation [6*,7-14,15*], inflammation [ 16,17*] and angiogenesis [ 18,191.

Qualitative

PCR for detection

of cytokine

gene

expression After amplification of cDNA (reverse-transcribed RNA) using specific primers for a particular cytokine, the product (the size of which is predicted by the position of the primers on the cDNA sequence) may be visualized on an agarose or acrylamide gel, stained with ethidium bromide. This signal is not easily quantifiable. The sensitivity of detection and specificity of the PCR-product can be further verified by probing samples transferred to a nylon membrane with a 32P-labelled internal oligonu-

Abbreviations CM-CSF-granulocyte

macrophage colony stimulating factor; IL-interleukin; Taq-Thermus

@ Current

aquaticus DNA

Biology

PCR-polymerase

chain reaction;

polymerase.

Ltd ISSN 0952-7915

211

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Immunological

techniques

cleotide (see below). This radioactive signal can be accurately quantitated either by densitometric measurement of autoradiographs, or by image scanning of the nylon membrane. This method has been used successfully to determine whether particular cytokines are expressed by different cell populations [7,8,10]. Although these studies provide important information on the expression of cy tokine mRNA it is not possible to make any quantitative statement for a number of reasons (see below). One key parameter is the possibility that different primers detecting different cytokines may have different sensitivities and thus it is not always possible to show that one cell population expresses one cytokine but not another. However, when there is reciprocal expression of cytokines by two populations of cells, it may be possible to draw the above conclusion. For example, Lowry and colleagues [20] using a murine model of transplantation have shown that allograft rejection is accompanied by the production of interleukin (IL)-2, but little IL-4, whereas during allograft acceptance there is production of IL-4 but little IL-2. This reciprocal expression of cytokines bypasses the need for strict quantitation, and the need for primers to have the same range of sensitivities. Similar data have been presented with regard to cytokine profiles in leprosy [ 15’1, although in this case the sensitivity of PCR was shown to be the same for each cytokine, and thus could be regarded as semi-quantitative.

Quantitation

of cytokine

using PCR amplification

mRNA expression of reverse-transcribed

RNA A number of parameters must be addressed in order to make any quantitative statement about mRNA expression using PCR. (a)

(b)

Comparison of mRNA expression for different cy tokines by the same and/or different cell populations requires the sensitivity of detection for each cytokine to be equivalent. This can be achieved by titration of plasmid cDNAs, and establishing that the ranges over which the primers for each respective cytokine can be detected are similar [IS-]. The sensitivity will rely on the elhcienq of reverse-transcription and specific PCR amplification for each cytokine. An alternative method (dependent on the availability of sufficient RNA) would be to ensure that mRNA detection by Northern blot analysis or by PCR amplification of reverse-transcribed RNA correlates closely between different cytokines, or between the same cytokine in different cell populations. The result should be standardized with an exogenous mRNA which is coreverse-transcribed and co-amplified in the same reaction as the transcripts under analysis. Whether the standard should be tested in parallel with the sample or

included in the same reaction tube is discussed below. The use of different types of standards is also discussed below and should be chosen to suit the needs of each particular study.

cc>The

number of cycles used for PCR amplification must be checked, to determine the range of detectability of a particular cytokine in a sample.

(4

This number of cycles must be chosen that the signal lies on the exponential standard curve.

Competitive

to ensure part of a

PCR using a cDNA standard

A method for quantitating granulocyte macrophagecolony stimulating factor (GM-CSF) and IL-3 mRNA was described by Gilliland et al. [17*]. A competitor DNA fragment, that differs from the cDNA of interest by having either a small intron or a mutated restriction enzyme site, is co-amplified with the sample in the same tube. Thus, the same primers are used to co-amplify the unknown and the competitor, and the sample can be distinguished from the standard. The two PCR products can then be easily separated by gel electrophoresis after amplification. Because the same primers are used, the cDNA and the standard DNA should be amplified with the same efficiency, provided that amplification of the standard DNA copy is unaffected by its small intron. By titrating an unknown amount of a cDNA template against a dilution series containing known amounts of the corresponding standard template, it should be possible to reliably and reproducibly quantitate the amount of cDNA present. Theoretically, this method provides a strategy for absolute and reproducible quantitation of cDNA by PCR. However, since it uses a DNA standard, there is no standardization of reverse-transcription of the sample RNA into cDNA.

Standardization PCR amplification

of reverse-transcription

and

for mRNA analysis

Quantitation of specific mRiiA by PCR has also been achieved using a synthetic cRNA as an internal standard [I(,]. The specific target mRNA and the cRNA standard are rc~t’rse-transcribed and co-amplified in the same tuhe. The same PCR primers are used, but each yields a PCR.product of a different size. The two PCR products can then be easily separated by gel electrophoresis after amplification. The amount of mRNA is then quantitated by extrapolating against the standard curve generated with the internal standard cRNA. The synthetic cRNA standard consists of a linear array of the sequences of upstream primers of multiple target genes followed by the sequences complementary to their downstream primers, in the same order. In the exponential phase of the amplification, the amount of target mRNA can be quantitated

PCR for detection

by extrapolating against the standard curve. In addition, the same internal standard cRNA can be used, with appropriate primer pairs, to quantitate multiple different mRNA species in a sample with < 0.1 ng of total RNA [ I6*]. By reverse transcription and amplification of the target mRNA and the internal standard cRNA in the same tube, variable effects due to differences in sample preparation, conditions of reverse transcription, or PCR amplification are internally controlled [ 161. It has been shown that the amount of amplified DNA fragment in a given sample has a prevailing influence on the efficiency of the PCR [ 161. When a high template concentration is used or occurs as a result of the PCR amplifications, phenomena such as substrate saturation of enzyme, product inhibition of enzyme, incomplete strand separation, and product strand reannealing can be limiting factors for efficient amplification. Thus, in order for the standard to appropriately control the target mRNA, the range of concentrations for both templates, as well as the number of amplification cycles should be such that the reactions remain within the exponential phase. This requires titration of the sample and standard mRNA or, when using the previous competitive PCR method, titration of the sample cDNA and standard cDNA, making analysis of one sample a lengthy, although accurate procedure. Furthermore, although using cRNA as an internal standard is effective in standardizing. the reverse transcription and PCR reactions, it relies on being able to get an accurate quantitation of the input RNA whichis not always possible when obtaining RNA from small numbers of cells.

Semi-quantitative expression

of cytokine

gene expression

O’Garra

and Vieira

ing RNA recovery as well as a standardization of the subsequent reverse transcription and PCR reactions, various researchers have made use of a house-keeping enzyme, such as actin, DHFR or HPRT as an internal standard [6*,7-10,12-14,15*,18,19]. These standards may not al ways be adequate since they may be expressed at much higher levels than the cytokine RNA and thus do not show up those differences which may have been obvious for messages with lower copy numbers. It is essential that the range of sensitivity of the primers used for detecting the house-keeping enzyme, is close to cytokine mRNA detection range [6-l, and that reactions yield results for both internal standard and test cytokine which lie within the exponential phase of the detection curve. If a uniform population of cells is being analysed, an alternative to a house-keeping enzyme, the amount of which may vary according to the activation state of the cell, is to use a cell marker, which may fluctuate less. An example is the use of the Ca chain as an internal standard for measurement of VP usage by PCR [21]. Alternatively, a cell line from a different species may be added to the cells in which cytokine expression is being studied, and the target cytokine level related back to expression of a particular gene expressed by that cell line ( [22], R Coffman, personal communication). Such a standard may serve as a carrier, in addition to providing a measure of the recovery , of RNAfrom small numbers of cells. As with some housekeeping enzymes, it may be expressed at a higher level than the cytokine gene and thus not give a true meas ure of fluctuations which may occur in RNApreparations. Thus, the use of internal standards is limited to those that are expressed at a similar detection range as cytokines.

PCR for the study of mRNA

from a small number

of cells

It is not always possible to quantitate the amount of RNA obtained from a small number of cells, either because of low yields, or because carrier RNA was added to ensure good recoveries. Thus, the results obtained after reverse transcription and PCR amplification for a particular cytokine must often be related back to input cell number. In order to cater for differential losses dur-

Simplification

of PCR product

enable handling

detection to

of a large number

of samples

In order to facilitate the handling of large numbers of samples, it is often adequate to run a standard curve of a positive control (known amounts of input RNA) in parallel with the test samples for reverse transcription and PCR, to which the test sample can be related. Although this will only give relative levels of specific RNA content

Fig. 1. Design strategy for primers probe. Primers to

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213

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Immunological

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this is usually sufficient for the needs of many studies, and not only serves to standardize the reaction but to ensure that the test samples lie within the exponential phase of a standard curve. It is advisable to include replicates of the standard curve and the sample under test and to repeat these a number of times to ensure reproducibility [6-l. An alternative to visualizing PCR products on a gel and subsequent probing of samples transferred to a nylon membrane with a QP-labeled internal oligonucleotide is to directly apply the PCR product to a nylon ftlter using a dot-blot apparatus, and then probe [6-,131. The radioactive signal is then quantitated either by densitometry of autoradiographs or by image scanning of the membrane. The dot-blot method facilitates the assay of a large number of samples. However, it cannot be used when there is a need to distinguish an internal standard on the basis of size (e.g. competitive PCR), and requires the use of PCR primers and probes which will not detect genomic DNA The use of the new Perkin Elmer Thermal Cycler 9600, enables handling of large numbers of samples since it has a tube format which enables the use of multi-channel pipettes.

sequence. The most efficient reverse transcription is generally achieved towards the 3’ end of the coding region of the mRNA sequence [ 51. In cases where the genomic structure is known, one can design each primer to span an intron-exon border, as depicted in Fig. 1. This will be an advantage because the presence of an intron with little or no sequence homology to the primer renders the PCR reaction very inefficient at amplifying genomic DNA, although at a low stringency of annealing, amplification of a genomic product of a different size may occur. As depicted schematically in Fig. 2, the use of an internal probe that also spans an intron-exon boundary when used at high stringency and validated by DNA dot-blot analysis detects the product of cDNA but not genomic DNA, amplification.

Conclusions The use of PCR for amplification of reverse-transcribed RNA (tDNA) has facilitated the study of cytokine expression in normal cells which has previously been limited by small numbers of cells and low mRNA expression. This extremely powerful tool is invaluable for the detailed study of immune and inflammatory responses.

Acknowledgements We should like to thank Daniel Finn for his help with compilation of the manuscript, DNAX Research Institute is supported by Schering Plough Corporation.

References

and recommended

Papers of particular interest, published view, have been highlighted as: of special interest . .. of outstanding interest

Fig. 2. Design strategy

for primers and probe. (a) Low level amplification of contaminating genomic DNA can be detected by ethidium bromide staining. (b) A radio-labelled internal probe designed as in Fig. 1, when used at high stringency, will detect cDNA amplification but not genomic DNA.

Primer and probe design for PCR There are a number of guidelines for the design of primers for PCR amplification for the detection of mRNA, which should be considered. Target mRNA sequences for different cytokines should be a similar size (0.2 to 0.5 kb in length), and positioned at similar parts of the coding

reading

within the annual period of re-

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A O’Garra and P Vieira, DNAX Research Institute, 901 California Avenue, Palo Alto, California 94304, USA

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