 RT-PCR & real-time RT-PCR
applications in Molecular
Immunology
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.
Hopkins J. Vet Immunol Immunopathol 2002 Sep 10;87(3-4):245-249
Department of Veterinary Pathology, Royal (Dick) School of
Veterinary Studies, 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. 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.
(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.
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
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.
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.
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
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.
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,
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.
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, 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.
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, 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.
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.
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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