RT-PCR  &  real-time RT-PCR applications in Molecular Immunology

  • Molecular Immunology  (general topics)

Nucleic acid approaches for detection and identification of biological warfare
and infectious disease agents

Dmitri Ivnitski1, Daniel J. O’Neil2, Anthony Gattuso3, Roger Schlicht3, Michael Calidonna4, and Rodney Fisher4
BioTechniques 35:862-869 (October 2003)

Biological warfare agents are the most problematic of the weapons of mass destruction and terror. Both civilian and military sources predict that over the next decade the threat from proliferation of these agents will increase significantly. In this review we summarize the state of the art in detection and identification of biological threat agents based on PCR technology with emphasis on the new technology of microarrays. The advantages and limitations of real-time PCR technology and a review of the literature as it applies to pathogen and virus detection are presented. The paper covers a number of issues related to the challenges facing bio-logical threat agent detection technologies and identifies critical components that must be overcome for the emergence of reliable PCR-based DNA technologies as bioterrorism countermeasures and for environmental applications. The review evaluates various system components developed for an integrated DNA microchip and the potential applications of the next generation of fully auto-mated DNA analyzers with integrated sample preparation and biosensing elements. The article also reviews promising devices and technologies that are near to being, or have been, commercialized.

 Molecular Immunology - Gene regulation and signal transduction
Hopkins J.
Vet Immunol Immunopathol  2002 Sep 10;87(3-4):245-249

Department of Veterinary Pathology, Royal (Dick) School of Veterinary Studies,
University of Edinburgh, Summerhall, Edinburgh EH9 1QH, UK.

Research on 'molecular immunology-gene regulation and signal transduction' in veterinary species is relatively new. The reason for its novelty is that until recently there have been very few tools with which we can work. Over the last 10 years the veterinary immunology community has succeeded in generating panels of defined monoclonal antibodies (mAb) and cloned genes that has enabled such work

to be started. More recently, quantitative, high-resolution analytical tools for veterinary species have begun to be developed; some of these are specific for veterinary species and others have been adapted from human or rodent systems. Of the species-specific tools that have recently been developed perhaps the most widely used are the immunoassays for cytokines, RNAase protection assays (RPAs) nd in the near future oligonucleotide and EST-based microarrays. This presentation will describe some of these assays and discuss their relative advantages and disadvantages.

Involvement of Pro-Inflammatory Cytokines, Mediators of Inflammation, and Basic Fibroblast Growth Factor in Prostaglandin F2a-Induced Luteolysis in Bovine Corpus Luteum
T.P. Neuvians, D. Schams,2 B. Berisha, and M.W. Pfaffl
Department of Physiology, Technical University Munich, Weihenstephaner Berg 3,
D-85350 Freising-Weihenstephan, Germany
BIOLOGY OF REPRODUCTION 70, 473–480 (2004)

The process of luteolysis requires very subtly modulated coordination of different factors and regulation systems. Immune
cells and cytokines were shown to be relevant for bovine luteolysis. The aim of this study was to investigate the detailed pattern
of mRNA expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNFalpha), TNF receptor type 1 (TNF-R1), interleukin 1beta (IL-1beta), and interferon gamma (IFNgamma), and of the inducible nitric oxide synthase (iNOS) and the basic fibroblast growth factor (FGF-2) during prostaglandin (PG) F2alpha-induced luteolysis in the bovine corpus luteum (CL). In addition, the mRNA expression for the LH-receptor (LH-R) and the steroidogenic enzyme P450scc was determined. Cows in the midluteal phase (Days 8–12) were injected with the PGF2alpha analogue cloprostenol, and CL were collected by transvaginal ovariectomy before and 2, 4, 12, 48, and 64 h after PGF2alpha injection. Conventional and real-time reverse transcription polymerase chain reaction RT-PCR (LightCycler) using SYBR Green I detection were employed to determine the mRNA expression for the investigated factors. All cytokines were significantly up-regulated during induced luteolysis. LH-R and P450scc mRNA were down-regulated ( P<0.05) during structural luteolysis (after 12 h), and P450scc in addition at 2 h after PGF2a ( P < 0.05). FGF-2 expression increased ( P<0.001) during functional luteolysis (until 12 h after PGF2alpha) and diminished thereafter. The mRNA expression for iNOS decreased ( P<0.05) after induction of luteolysis. In conclusion, cytokines may be involved not only in structural but also in functional luteolysis and the deprivation of luteal survival factors, leading to a situation where apoptosis can occur. FGF-2 may participate in the suppression of cytokine-induced iNOS mRNA expression and in the prevention of an inflammatory reaction in the surrounding tissues.

Rat pro-inflammatory cytokine and cytokine related mRNA quantification
by real-time polymerase chain reaction using SYBR green

André Peinnequin*1, Catherine Mouret1, Olivier Birot2, Antonia Alonso1,
Jacques Mathieu1, Didier Clarençon1, Diane Agay1, Yves Chancerelle1 and
Eric Multon1
BMC Immunology 2004, 5:3

Background: Cytokine mRNA quantification is widely used to investigate cytokine profiles,
particularly in small samples. Real-time polymerase chain reaction is currently the most reliable
method of quantifying low-level transcripts such as cytokine and cytokine receptor mRNAs. This
accurate technique allows the quantification of a larger pattern of cytokines than quantification at
the protein level, which is limited to a smaller number of proteins.

Results: Although fluorogenic probes are considered more sensitive than fluorescent dyes, we
have developed SYBR Green real-time RT-PCR protocols to assay pro-inflammatory cytokines
(IL1a, IL1b and IL6, TNFa), cytokine receptors (IL1-r1, IL1-r2, IL6-r, TNF-r2) and related molecules
(IL1-RA, SOCS3) mRNA in rats. This method enables normalisation against several housekeeping
genes (beta-actin, GAPDH, CypA, HPRT) dependent on the specific experimental treatments and
tissues using either standard curve, or comparative CT quantification method. PCR efficiency and
sensitivity allow the assessment of; i) basal mRNA levels in many tissues and even decreases in
mRNA levels, ii) mRNA levels from very small samples.

Conclusion: Real-time RT-PCR is currently the best way to investigate cytokine networks. The
investigations should be completed by the analysis of genes regulated by cytokines or involved in
cytokine signalling, providing indirect information on cytokine protein expression.

 An overview of real-time quantitative PCR: applications 
to quantify cytokine gene expression

Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C. (2001)

Methods  2001 Dec;25(4):386-401 

The analysis of cytokine profiles helps to clarify functional properties of immune cells, both for research and for clinical diagnosis. The real-time reverse transcription polymerase chain reaction (RT-PCR) is becoming widely used to quantify cytokines from cells, body fluids, tissues, or tissue biopsies. Being a very powerful and sensitive method it can be used to quantify mRNA expression levels of cytokines, which are often very low in the tissues under investigation. The method allows for the direct detection of PCR product during the exponential phase of the reaction, combining amplification and detection in one single step. In this review we discuss the principle of real-time RT-PCR, the different methodologies and chemistries available, the assets, and some of the pitfalls. With the TaqMan chemistry and the 7700 Sequence Detection System (Applied Biosystems), validation for a large panel of murine and human cytokines and other factors playing a role in the immune system is discussed in detail. In summary, the real-time RT-PCR technique is very accurate and sensitive, allows a high throughput, and can be performed on very small samples; therefore it is the method of choice for quantification of cytokine profiles in immune cells or inflamed tissues.

Relative quantification of mRNA levels in Jurkat T cells with RT-real time-PCR
(RT-rt-PCR): new possibilities for the screening of anti-inflammatory and cytotoxic compounds

Gertsch J, Guttinger M, Sticher O, Heilmann J.
Pharm Res  2002 Aug;19(8):1236-43

Swiss Federal Institute of Technology (ETH) Zurich, Institute of Pharmaceutical Sciences.

Purpose. Quantification of the pro-inflammatory action of mitogens on mRNA levels of growth-related genes, transcription factors, and cytokines in T cells as markers for the screening of compounds with immunomodulatory, anti-inflammatory or cytotoxic potential.
Method. A reverse transcription-real time-polymerase chain reaction assay with TaqMan probes was developed. Jurkat T cells were treated with cyclosporin A, hypericin, capsaicin, and catechin before phorbol 12-myristate 13-acetate stimulation, and their effects on the relative mRNA levels were determined. A cell viability assay was performed in parallel. 
Results. Cyclosporin A and capsaicin were potent inhibitors of PMAinduced cytokine transcription. Cyclosporin A further targeted cyclin D1 transcription. Capsaicin exhibited no effects on the cell viability at low concentrations, whereas cyclosporin A did. Hypericin downregulated nearly all investigated mRNAs, resulting in a strong timedependent cytotoxicity. Catechin showed no effects on mRNA levels and cell viability. 
Conclusions. The inhibition of the up-regulation of mRNA levels of cytokines points to a specific anti-inflammatory potential of capsaicin. Hypericin showed no specific effects on the mRNA expression. The overall decrease of mRNA levels is probably an early indication of the strong cytotoxic effect observed after 48 h. Therefore, quantification of mRNA levels by reverse transcription-real timepolymerase chain reaction is, in combination with the monitoring of cell viability, a valuable tool to distinguish between specific immunomodulatory and cytotoxic effects in vitro.

Cytokine mRNA quantification by real-time PCR
Stordeur P, Poulin LF, Craciun L, Zhou L, Schandene L, de Lavareille A, Goriely
S, Goldman M.
J Immunol Methods  2002 Jan 1;259(1-2):55-64

Departement d'Immunologie-Hematologie-Transfusion, Hopital Erasme, Brussels,

Real-time PCR represents a new methodology that accurately quantifies nucleic acids. This has been made possible by the use of fluorogenic probes, which are presented in two forms, namely hydrolysis probes (also called TaqMan probes) and hybridisation probes. We decided to apply this methodology to cytokine mRNA quantification and this led us to the development of a protocol that provides an easy way to develop and perform rapidly real-time PCR on a Lightcycler instrument. It was made possible by the use of freely available software that permits a choice of both the hydrolysis probe and the primers. We firstly demonstrated that the reproducibility of the method using hydrolysis probes compares favourably with that obtained with hybridisation probes. We then applied this technique to determine the kinetics of IL-1ra, IL-1beta, IL-5, IL-13, TNF-alpha and IFN-gamma induction upon stimulation of human peripheral
blood mononuclear cells (PBMC) by phytohaemagglutinin (PHA). Finally, the method was also used successfully to demonstrate that IFN-alpha induces IL-10 mRNA accumulation in human monocytes.

Rapid quantitation of proinflammatory and chemoattractant cytokine expression in
small tissue samples and monocyte-derived dendritic cells: validation of a new
real-time RT-PCR technology

Blaschke V, Reich K, Blaschke S, Zipprich S, Neumann C.
J Immunol Methods  2000 Dec 1;246(1-2):79-90

Department of Dermatology, von-Siebold-Str. 3, D-37075, Goettingen, Germany

The analysis of cytokine profiles plays a central part in the characterization of disease-related inflammatory pathways and the identification of functional properties of immune cell subpopulations. Because tissue biopsy samples are too small to allow the detection of cytokine protein, the detection of mRNA by RT-PCR analysis is often used to investigate the cytokine milieu in inflammatory lesions. RT-PCR itself is a qualitative method, indicating the presence or absence of specific transcripts. With the use of internal or external standards it may also serve as a quantitative method. The most widely accepted method is quantitative competitive RT-PCR, based on internal shortened standards. Recently, online real-time PCR has been introduced (LightCycler), which allows quantitation in less than 30 min. Here, we have tested its use for the analysis of cytokine gene expression in different experimental in vitro and ex vivo settings. First, we compared quantitative competitive RT-PCR with real-time RT-PCR in the quantitation of transcription levels of the CD4(+) cell-specific chemoattractant Interleukin-16 during the maturation of monocyte-derived dendritic cells, and found a good correlation between both methods. Second, differences in the amounts of IL-16 mRNA in synovial tissue from patients with rheumatoid arthritis and osteoarthritis as assessed by real-time RT-PCR paralleled differences in the level of IL-16 protein in the synovial fluid. Finally, we employed real-time RT-PCR to study the cutaneous expression of several cytokines during experimental immunomodulatory therapy of psoriasis by Interleukin-10, and demonstrate that the technique is suitable forpharmacogenomic monitoring. In summary, real-time RT-PCR is a sensitive and rapid tool for quantifying mRNA expression even with small quantities of tissue. The results obtained do not differ from those generated by quantitative competitive RT-PCR.

   Quantitative monitoring of the mRNA expression pattern of the TGF-beta-isoforms (beta-1, beta-2, beta-3) during transdifferentiation of hepatic stellate cells  using a newly developed real-time SYBR Green PCR
L. Wickert, , S. Steinkrüger, M. Abiaka, U. Bolkenius, O. Purps, C. Schnabel and A. M. Gressner
Biochem Biophys Res Commun 2002 Jul 12;295(2): 330-335

Institute of Clinical Chemistry and Pathobiochemistry, RWTH-University Hospital Aachen

   Current methods to determine the mRNA of the TGF-beta-isoforms, beta-1, beta-2,  and beta-3, are not sensitive enough to detect small alterations in the expression levels. Therefore, we established a SYBR Green I-based real-time quantitative PCR procedure with fragment-specific standards. The advantage of gene-specific quantification is the possibility to be abstain from the need to compare results with a house-keeping gene having a different sequence and PCR efficiency. Reproducibility of the results and analytical variances of the real-time PCR assays were tested. In transdifferentiating rat hepatic stellate cells (HSC) the TGF-1-mRNA was  found to be the predominant isoform expressed followed by TGF-3 and low amounts of TGF-2-mRNA. An alteration of the TGF-1,-2, and -3 ratio during HSC transdifferentiation could not be detected. Furthermore, the GAPDH mRNA expression varied during HSC activation, and thus is not recommended as a standard in real-time PCR quantifications.

Quantitative real-time PCR for the measurement of feline cytokine mRNA
Leutenegger CM, Mislin CN, Sigrist B, Ehrengruber MU, Hofmann-Lehmann R, Lutz H.
Vet Immunol Immunopathol  1999 Nov 30;71(3-4): 291-305

Clinical Laboratory, Department of Internal Veterinary Medicine, University of
Zurich, Switzerland

We have developed real-time PCR systems to quantitate feline cytokine gene expression. The method is based on the cleavage of fluorescent dye-labelled probes by the 5'-3' exonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by a Sequence Detection System. The feline-specific TaqMan probes were designed to encompass an intron, thus allowing differentiation of complementary DNA versus genomic DNA amplification products. Quantitative analysis of cytokine cDNA concentrations was performed in comparison to feline GAPDH. Messenger RNA (mRNA) from the universally expressed housekeeping gene GAPDH proved to be useful as an amplification control and allowed for correction of variations in the efficiencies of RNA extraction and reverse transcription. GAPDH mRNAs were readily detectable in cDNAs prepared from unstimulated feline peripheral blood mononuclear cells (PBMCs) and from frozen cell pellets, while cytokines (Interleukin (IL)-4, IL-10, IL-12 p35, IL-12 p40, IFNgamma, IL-16) were expressed at variable amounts. IFNgamma transcription was found to be upregulated in stimulated PBMCs and feline cell lines. The synthesis of cDNA and the performance of the PCR in separate tubes proved to be of superior sensitivity compared to a single-tube based system. The assays described are highly reproducible, require no post-PCR manipulation of the amplicons and permit the analysis of several hundred PCR reactions per day. With this method it is possible to detect and quantify cytokine mRNA expression reliably in small amounts of cells even after storage of samples for at least 5 years.

Quantitation of bovine cytokine mRNA in milk cells of healthy cattle by
real-time TaqMan polymerase chain reaction

Leutenegger CM, Alluwaimi AM, Smith WL, Perani L, Cullor JS.
Vet Immunol Immunopathol  2000 Dec 29;77(3-4):275-87

Department of Medicine and Epidemiology, School of Veterinary Medicine,
University of California, Davis, CA 95616, USA

Here we present a novel methodology to quantitate bovine cytokines and growth factors contributing to immunity against bacterial infections of the mammary gland in cattle. Real-time TaqMan PCR systems were developed to overcome limitations of conventional quantitative PCR methods. The TaqMan method is based on the cleavage of fluorescent dye-labeled probes by the 5'-3' exonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by an automated spectrophotometer integrated in a sequence detection system (Applied Biosystems, Foster City, CA). The bovine-specific TaqMan probes were designed to encompass an intron, thus allowing differentiation between complementary DNA (cDNA) and genomic DNA (gDNA) amplification products. Quantitative analysis of cytokine cDNA was performed in comparison to bovine glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Messenger RNA (mRNA) from the universally expressed housekeeping gene GAPDH proved to be useful as an amplification control and allowed for correction of variations in different numbers of cells in the starting material, in the efficiencies of RNA extraction and reverse transcription. With this method, high-throughput analysis of large numbers of samples was possible within a short time. In addition, decreasing the numbers of working steps shortened the time for analysis and increased accuracy. Profiles of cytokines (interleukin (IL)-2, IL-6, IL-8, IL-12 p40, TNF-alpha, IFN-gamma) and granulocyte-macrophage colony stimulating factor (GM-CSF) were established in normal lactating cattle. Differences of cytokine profiles obtained with the real-time TaqMan PCR system and conventional methods are discussed.

Quantification of murine cyctokine mRNAs using real-time q-RT-PCR
Lut Overbergh, Dirk Valckx, Mark Waer and Chantal Mathieu
Cytokine 11(4): 305-312 (1999)

Laboratory for Experimental Transplantation, U.Z.Gasthuisberg, Herestraat 49, Catholic University of Leuven, 3000,
Leuven, Belgium

Recently, a novel technique for "real time" quantitative Reverse Transcriptase-PCR which measures PCR-product accumulation during the    exponential phase of the PCR reaction using a dual-labelled fluorogenic probe, has been developed. This method allows direct detection of PCR-product formation by measuring the increase in fluorescent emission continuously during the PCR reaction. Here we present data validating this PCR-method for the quantification of murine cytokines and other factors playing a role in immune regulation (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p40, IL-13, IL-15, IFN-TNF-TGF- and iNOS). For each substance of interest, a set of primers and internal probe was designed, which specifically amplify the target cDNA, not co-amplifying contaminating genomic DNA. Furthermore, a corresponding reference plasmid cDNA clone was constructed, allowing direct quantification. Additionally, normalization to the housekeeping genes -actin or GAPDH was performed. The assay is very sensitive and accurate. It is a "closed-tube" PCR reaction, avoiding time-consuming and hazardous post-PCR manipulations and decreasing the potential risk of PCR contamination.

Increased expression of pro-inflammatory cytokines and lack of up-regulation of
anti-inflammatory cytokines in early distemper CNS lesions

Markus S, Failing K, Baumgartner W.
J Neuroimmunol  2002 Apr;125(1-2):30-41

Institut fur Veterinar-Pathologie, Justus-Liebig-Universitat Giessen,
Frankfurter Str. 96, 35392 Giessen, Germany.

To investigate the pathogenesis of early lesions in canine distemper virus (CDV) leukoencephalomyelitis, the expressions of pro- and anti-inflammatory cytokines such as interleukin (IL)-1beta, IL-2, IL-6, IL-8, IL-10, IL-12, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma and transforming growth factor (TGF)-beta and the housekeeping genes beta-actin and GAPDH were studied using semi-quantitative RT-PCR. Relative cytokine values were related to the degree of CDV infection, MHC class II expression and infiltration of CD4-, CD8- and CD3epsilon-positive lymphocytes. Actin up-regulation, in contrast to GAPDH, was influenced by CDV infection and therefore could not be used as an internal standard to study cytokine expression. In early CDV infection of the cerebellum, either no detectable lesions or mild infiltration of CD8 positive cells or demyelination and up-regulation of MHC class II antigen were observed. IL-6, -8, -12 and TNF-alpha transcripts were found in 94%, 94%, 78% and 56% of distemper dogs, respectively, compared to 17%, 33%, 0% and 0% in controls, whereas IL-1beta, -2 and IFN-gamma were not detectable in any of the studied cerebella. Conversely, IL-10 and TGF-beta transcripts were present in 83% and 100% of the investigated cerebella of distemper dogs and controls. Relative RT-PCR results, expressed as %GAPDH, revealed a significant up-regulation of IL-6, -8, -12 and TNF-alpha mRNA in distemper dogs; whereas IL-10 and TGF-beta showed only a weak and not significantly increased expression following infection. Relative pro-inflammatory cytokine expression values were highest following CDV infection, indicating that the virus itself directly triggered the up-regulation of the pro-inflammatory cytokines. Succeeding changes, such as lymphocyte infiltration, MHC class II up-regulation and demyelination resulted only in a minor additional increase in cytokine expression, implying a secondary or by-stander mechanism of cytokine activation by these changes. Disease initiation and progression in early distemper leukoencephalomyelitis seemed to be due to a lacking or inappropriate response of the anti-inflammatory cytokines in the presence of a vigorous up-regulation of pro-inflammatory cytokines.

A reverse transcription-polymerase chain reaction method to analyze porcine
cytokine gene expression

Dozois CM, Oswald E, Gautier N, Serthelon JP, Fairbrother JM, Oswald IP.
Vet Immunol Immunopathol  1997 Sep 19;58(3-4):287-300

Laboratoire de Pharmacologie Toxicologie, INRA, Toulouse, France.

A reverse transcription-polymerase chain reaction (RT-PCR) method was developed in order to provide a highly sensitive, rapid, and simple means of simultaneously measuring the expression of porcine cytokines in immune cell populations. Oligonucleotide primers were designed to amplify porcine cytokine cDNA from genes encoding IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFN-gamma, TNF-alpha, TNF-beta and the housekeeping genes beta-actin and cyclophilin by PCR. Primers were chosen from different exons to detect for possible genomic DNA contamination of samples. To validate RT-PCR, unstimulated and concanavalin A (ConA) stimulated porcine peripheral blood mononuclear cells (PBMCs) were cultured from 2 h to 72 h, RNA was extracted and reverse transcribed, and cDNA was amplified using the different primer sets. Band intensities of PCR products were quantified by densitometric scanning and values were normalized against cyclophilin. For each of the cytokines, the kinetics of gene expression were similar among PBMCs isolated from different animals and could be grouped into two main patterns. Lymphocyte derived cytokines (IL-2, IL-4, IFN-gamma, and TNF-beta) exhibited low level expression in unstimulated cells and increased expression in ConA-stimulated PBMCs. IFN-gamma and IL-2 mRNA levels peaked at 24 h and returned to baseline by 72 h, whereas IL-4 and TNF-beta mRNA levels did not return to baseline by 72 h. In contrast, substantial mRNA levels for inflammatory cytokines (IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-12, and TNF-alpha) and IL-10 were detected from both unstimulated and ConA-stimulated PBMCs. Results indicate that RT-PCR is a sensitive and convenient method to monitor cytokine mRNA expression in porcine samples.

Differential expression of IFN-a subtypes in human PBMC: evaluation of novel real-time PCR assays
S. Lo¨seke*, E. Grage-Griebenow, A. Wagner, K. Gehlhar, A. Bufe (2003)
Ruhr-University Bochum, Experimental Pneumology, University Hospital Bergmannsheil, BGFA XU 19,
Bu¨rkle-de-la-Camp-Platz 1, D-44789 Bochum, Germany

Studies of the human IFN-a subtype system have been hampered by the lack of efficient procedures to quantify and differentiate the expression of the highly homologous IFN-a subtypes. Here we evaluate four novel real-time PCR assays for the specific detection and quantification of IFN-a mRNA for the subtypes a2, a6, a8 and a1/13 in a combined assay in human peripheral blood mononuclear cells (PBMC). This included (a) the selection of h-glucuronidase (GUS) as a suitable housekeeping gene for relative quantification; (b) verification of the specificity by using human DNA of different IFN-a subtypes; and (c) comparison of the amplification efficiencies among the different assays. This highly sensitive method allows the detection of low-level, constitutive IFN-a mRNA and shows differences in the composition of constitutive IFN-a subtypes compared to other cell types (HeLa and HEp-2). The in vitro stimulation of PBMC with Newcastle disease virus (NDV), Respiratory syncytial virus (RSV) or an inactivated Herpes simplex (HSV) preparation leads to the transcriptional induction of all IFN-a subtypes investigated but to different expression levels. Among the subtypes detected, IFN-a13/1 and a2 are the major transcripts followed by a8, and finally a6 as a minor transcribed subtype. Time-kinetics of IFN-a transcriptional activation also revealed variations in the course of IFN-a transcription between NDV, RSVor HSV. The data obtained from the real-time PCR assays correlated well with IFN-a2 protein release. In conclusion, we have demonstrated the suitability and reliability of new real-time PCR assays for the rapid and efficient analysis of IFN-a subtype expression.

The Usefulness of Quantitative Real-Time PCR in Immunogenetics

Paurizio Provenzano, MD,1 Carlo Riccardo Rossi, MD,2 Simone Mocellin, MD1,2
Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD and
Department of Oncological and Surgical Sciences, Istituto di Clinica Chirurgica II,
University of Padova, Padova, Italy

The Use of Real-Time RT-PCR for the Quantification of Cytokine Gene Expression
L. Overbergh, A. Giulietti, D. Valckx, B. Decallonne, R. Bouillon, and C. Mathieu
J Biomol Tech 2003;14:33–43

Laboratory of Experimental Medicine and
Endocrinology (LEGENDO), Catholic University
of Leuven, U.Z. Gasthuisberg, Leuven, Belgium

Real-time reverse transcriptase polymerase chain reaction (RT-PCR) is becoming a widely used method to quantify
cytokines from cells, tissues, or tissue biopsies.The method allows for the direct detection of PCR product during the
exponential phase of the reaction, combining amplification and detection in a single step. Using TaqMan chemistry (Applied
Biosystems, Foster City, CA) and the ABI Prism 7700 Sequence Detection System (Applied Biosystems), we validated
a large panel of murine and human cytokines, as well as other factors playing a role in the immune system, such as
chemokines and apoptotic markers. Although the method allows fast, sensitive, and accurate quantification, different
control assays are necessary for the method to be reliable. By construction of complementary DNA (cDNA) plasmid
clones, standard curves are generated that allow direct quantification of every unknown sample. Furthermore, the choice
of a reliable housekeeping gene is very important. Finally, coamplification of contaminating genomic DNA is avoided by
designing sets of primers located in different exons or on intron–exon junctions. In conclusion, the real-time RT-PCR
technique is very accurate and sensitive, allows high throughput, and can be performed on very small samples.The development
of real-time RT-PCR has resulted in an exponential increase in its use over the last couple of years, and the
method has undoubtedly become the standard for quantifying cytokine patterns, clarifying many functional properties of
immune cells and their associated diseases.

Quantification of Cytokine Gene Expression Using an Economical real-time PCR
Method Based on SYBR Green I

R. Ramos-Payan, M. Aguilar-Medina, S. Estrada-Parra, J. A. Gonzalez-y-Merchand,z L. Favila-Castillo, A. Monroy-Ostria
& I. C. E. Estrada-Garcia*
Scandinavian Journal of Immunology 57, 439–445

Assessment of cytokine expression has become crucial to understand host
responses to infections as well as autoimmunity. Several approaches including
Northern blot, RNase protection assay and enzyme-linked immunosorbent assay
have been used for this purpose, but they are time consuming, labour intense,
and relatively large quantity of the samples is usually required. Recently, a
technique termed real-time reverse transcriptase-polymerase chain reaction
(RT-PCR) has been developed to determine genetic expression with great
sensitivity and specificity; however, specialized instrumentation and costly
reagents are usually needed. We aimed at using low-cost reagents for real-time
PCR. This was achieved by adapting a conventional RT-PCR protocol to the
quantitative real-time format, by the addition of the SYBR1 Green I reagent.
We validated the approach by assessing the cytokine gene expression of murine
splenocytes upon stimulation with phorbol 12-myristate 12-acetate (PMA)–
ionomycin. The results using this technique were compared with those obtained
with the well-established gene array method. We conclude that the use of the
SYBR1 Green I reagent during real-time RT-PCR provides a highly specific and
sensitive method to quantify cytokine expression with accuracy and no post-
PCR manipulation.

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