Absolute quantification in real-time RT-PCR

Calibration curves are highly reproducible and allow the generation of highly specific, sensitive and reproducible data. However, the external calibration curve model has to be thoroughly validated as the accuracy of absolute quantification in real-time RT-PCR depends entirely on the accuracy of the standards. Standard design, production, determination of the exact standard concentration and stability over long storage time is not straightforward and can be problematic. The dynamic range of the performed calibration curve can be up to nine orders of magnitude from <101 to >1010 start molecules, depending on the applied standard material. The calibration curves used in absolute quantification can be based on known concentrations of DNA standard molecules, e.g. recombinant plasmid DNA (recDNA), genomic DNA, RT-PCR product, commercially synthesized big oligonucleotide. Stability and reproducibility in kinetic RT-PCR depends on the type of standard used and depends strongly on ‘good laboratory practice’. Cloned recDNA and genomic DNA are very stable and generate highly reproducible standard curves even after a long storage time, in comparison to freshly synthesized RNA. Furthermore, the longer templates derived from recDNA and genomic DNA mimic the average native mRNA length of about 2 kb better than shorter templates derived from RT-PCR product or oligonucleotides. They are more resistant against unspecific cleavage and proofreading activity of polymerase during reaction setup and in kinetic PCR (own unpublished results). One advantage of the shorter templates and commercially available templates is an accurate knowledge of its concentration and length. A second advantage is that their use avoids the very time consuming process of having to produce standard material: standard synthesis, purification, cloning, transformation, plasmid preparation, linearization, verification and exact determination of standard concentration.

A problem with DNA based calibration curves is that they are subject to the PCR step only, unlike the unknown mRNA samples that must first be reverse transcribed. This increases the potential for variability of the RT-PCR results and the amplification results may not be strictly comparable with the results from the unknown samples. However, the problem of the sensitivity of the RT-PCR to small variations in the reaction setup is always lurking in the background as a potential drawback to this simple procedure. Therefore, quantification with external standards requires careful optimization of its  precision (replicates in the same kinetic PCR run – intra-assay variation) and reproducibility (replicates in separate kinetic PCR runs – inter-assay variation) in order to understand the limitations within the given application.

A recombinant RNA (recRNA) standard that was synthesized in vitro from a cloned RT-PCR fragment in plasmid DNA is one option. However, identical RT efficiency, as well as real-time PCR amplification efficiencies for calibration curve and target cDNA must be tested and confirmed if the recDNA is to provide a valid standard for mRNA quantification. This is because only the specific recRNA molecules are present during RT and the kinetics of cDNA synthesis are not like those in native RNA (the unknown sample) that also contain a high percentage of natural occurring sub-fractions, e.g. ribosomal RNA (rRNA, ~80%) and transfer RNA (tRNA, 10-15%). These missing RNA sub-fractions can influence the cDNA synthesis rate and in consequence RT efficiency rises and calibration curves are then overestimated in gene quantification. To compensate for background effects and mimic a natural RNA distribution like in native total RNA, total RNA isolated from bacterial or insect cell lines, can be used. Alternatively commercially available RNA sources can be used as RNA background, e.g. poly-A RNA or tRNA, but they do not represent a native RNA distribution over all RNA sub-species. Earlier results suggest, that a minimum of RNA background is generally needed and that it enhances RT synthesis efficiency rate. Low concentrations of recRNA used in calibration curves should always be buffered with background or carrier RNA, otherwise the low amounts can be degraded easily by RNAses. Very high background concentrations had a more significant suppression effect in RT synthesis rate and in later real-time PCR efficiency.

No matter how accurately the concentration of the standard material is known, the final result is always reported relatively compared to a defined unit of interest: e.g. copies per defined ng of total RNA, copies per genome (6.4 pg DNA), copies per cell, copies per gram of tissue, copies per ml blood, etc. If absolute changes in copy number are important then the denominator still must be shown to be absolute stable across the comparison. This accuracy may only be needed in screening experiments (amount of microorganism in food), to measure the percentage of GMO (genetic modified organism) in food, to measure the viral load or bacterial load in immunology and microbiology. The quality of your gene quantification data cannot be better than the quality of your denominator. Any variation in your denominator will obscure real changes, produce artificial changes and wrong quantification results. Careful use of controls is critical to demonstrate that your choice of denominator was a wise one. Under certain circumstances, absolute quantification models can also be normalized using suitable and unregulated references or housekeeping genes (see Normalization).


New papers using absolute quantification:

Interesting papers using absolute quantification:

Validities of mRNA quantification using recombinant RNA and 
recombinant DNA external calibration curves in real-time RT-PCR

M. W. Pfaffl & M. Hageleit
Biotechnology Letters (2001) 23, 275-282

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Summary

Reverse transcription (RT) followed by polymerase chain reaction (PCR) is the technique of choice for analysing mRNA in extremely low abundance. Real-time RT-PCR using SYBR Green I detection combines the ease and necessary exactness to be able to produce reliable as well as rapid results. To obtain high accuracy and reliability in RT and real-time PCR a highly defined calibration curve is needed. We have developed, optimised and validated an Insulin-like growth factor-1 (IGF-1) RT-PCR in the LightCycler, based on either a recombinant IGF-1 RNA (recRNA) or a recombinant IGF-1 DNA (recDNA) calibration curve. Above that, the limits, accuracy and variation of these externally standardised quantification systems were determined and compared with a native RT-PCR from liver total RNA. For the evaluation and optimisation of cDNA synthesis rate of recRNA several RNA backgrounds were tested. We conclude that external calibration curve using recDNA is a better model for the quantification of mRNA than the recRNA calibration model. This model showed higher sensitivity, exhibit a larger quantification range, had a higher reproducibility, and is more stable than the recRNA calibration curve.

Introduction

Because of its high sensitivity, RT-PCR is increasingly used to quantify physiologically changes in gene expression. The RT-PCR quantification technique of choice depends on the target sequence, the expected range of the mRNA amount present in the tissue, the degree of accuracy required, and whether quantification needs tobe relative or absolute (Freeman et al. 1999). The externally standardised  RT-PCR with quantification on ethidium bromide stained gels followed by densitometry is widely used, but the degree of accuracy is limited and the quantification is more relative than absolute. For a fully quantitative measurement of low abundant gene expression only a few PCR methods are reliable. At present internally standardised, competitive RT-PCR (Becker-Andreé et al. 1989; Gilliland et al. 1990;  Siebert & Larrick 1992), externally standardised, real-time RT-PCR using SYBR Green I technology (Morrison et al. 1998; Wittwer & Garling 1991), or externally standardised real-time RT-PCR using specific fluorescence dye labelled hybridisationrobe(s) (Leutenegger et al. 1999; Loeffler et al. 2000; Winer et al. 1999), are suitable for sensitive quantification. Probe-based detection formats are based on fluorescence resonance energy transfer (FRET) (Wittwer et al. 1997) in the LightCycler system (Roche Diagnostics, Basel, Switzerland) or on reporter fluorescence primarily by Förster type energy transfer (Förster 1948; Lakowicz 1983) in the TaqMan system (PE Applied Biosystems, Foster City, USA). Only real-time RT-PCR with SYBR Green I technology combines the ease and necessary exactness to be able to produce reliable as well as rapid results. Because of  its external calibration curve this method has to be highly validated and the identical LightCycler PCR amplification efficiencies for calibration curve and target mRNA must be confirmed. 
In general two types of external calibration curves are possible. Type one based on a recombinant RNA (recRNA) standard target and the type two on a recombinant DNA (recDNA) target. Alternatively, only a purified RT-PCR  product can be used as calibration material, but the long term stability and the reproducibility of the calibration is not very satisfactory (data not shown). In case of using the calibration recRNA model both, standard recRNA and unknown sample RNA, underwent parallel RT and real-time PCR conditions. If recDNA is used as calibration model, the standard samples only underwent the real-time PCR conditions and the amplification results may differ from the RT-PCR results in the unknown samples.

In this paper these two models are investigated in the LightCycler real-time PCR system, using a multi-species RT-PCR for IGF-1. IGF-1 is considered to mediate the anabolic growth hormone actions in various tissues and species. During postnatal growth, IGF-1 stimulates protein synthesis and improves glucose utilisation (Simmen 1991). Above that, locally expressed IGF-1 is an important growth regulator acting in an auto- and paracrine manner (Thissen et al. 1994). The aim of this study was the comparison of  two types of calibration models, leading back on the identical gene target, as well as the optimisation and validation of these models using SYBR Green I fluorescence technology. Results were compared with a native RT-PCR from liver total RNA. Derived limits and accuracy of this methodology are described and optimisation strategies for LightCycler PCR are discussed to achieve accurate, reproducible and reliable results in the unknown samples RNA. 
Table:  Characterisation of real-time IGF-1 LightCycler PCR using either recRNA or ss recDNA external calibration curve in comparison with the native liver total RNA. Intra- and inter-assay variation of calibration curve models are mean values (n =4). 

n = 4 IGF-1 rek RNA
calibration curve 		IGF-1 rek DNA
calibration curve 		unknown
IGF-1 mRNA
start template IGF-1 recRNA  IGF-1 recDNA IGF-1 mRNA
PCR efficiency 1.77 1.93 1.89
detection limit 16 m 6 m 80 pg liver total RNA
quantification limit 1600 m 60 m 500 pg liver total RNA
quantification range
(test linearity)		 1600 - 1.6 x 1010 m
(r = 0.992)		 60 – 6 x 1010 m
(r = 0.996)		 500 pg - 50 ng total RNA
(r = 0.933)
intra-assay variation 2.7% (n = 4)  0.7% (n = 4)  1.2% (n = 4)
inter-assay variation 4.5% (n = 4) 2.6% (n = 4) 4.9% (n = 4)

m = start molecules

Figure: Logarithmic fluorescence plot versus cycle number is shown, resulting from the determination of intra-assay variation in recDNA calibration curve model. Four replicates of different concentrations of recDNA start molecules were plotted [6 x 106 to 60   start molecules]. Analysis line was set to a fluorescence level of 2 where measurement of crossing points (CP) was done. Corresponding CP mean data are summarized in table above in column of intra-assay variation of recDNA.


 

Conclusion

The sensitivity, linearity and reproducibility of the developed real-time PCR assays allows for the absolute and accurate quantification of IGF-1 mRNA molecules even in tissues with low abundancies down to a few molecules. Using a recDNA calibration curve model only the existing cDNA molecules derived out of RT can be quantified precisely. It is not possible to draw a conclusion on the existing mRNA molecules present in the native total RNA sample. Always the cDNA synthesis efficiency must be recognised. Using a recRNA calibration curve model the advantage is that both RNA templates underwent parallel a RT and real-time PCR. But in this approach the following real-time PCR efficiency is suppressed and yields in a sub-optimal real-time quantification. In our opinion the external calibration curve using recDNA is a better model for the quantification of mRNA than the recRNA calibration model – consideration of RT efficiency is needed. The model showed higher sensitivity, exhibit a larger quantification range, had a higher reproducibility, and is more stable than the recRNA. We have used this recDNA system to compare the IGF-1 expression rates in bovine (Bos taurus) [EMBL Ac. no. X15726]  (Pfaffl et al. 1998b) and porcine tissues (Sus scrofa) [EMBL Ac. no. X17492] (Pfaffl et al. 1998a). It can also be used in other species like sheep (Ovis aries) [EMBL Ac. no. M30653] and primates (Callithrix jacchus) [EMBL Ac. no. Z49055] (Pfaffl 2000) with sufficiently high homologies of the amplified IGF-1 fragment.

 

High-Resolution Semi-Quantitative Real-Time PCR without the Use of a Standard Curve.
Gentle et al. 2001
BioTechniques 31:502-508 (September 2001)

The repeatability and sensitivity of a simple, adaptable, semi-quantitative, realtime RT-PCR assay was investigated. The assay can be easily and rapidly applied to quantitate relative levels of any gene product without using standards, provided that amplification conditions are specific for the PCR product of interest. Using the Light-Cycler‘ real-time PCR machine, a serial 10-fold dilution series (spanning four orders of magnitude) of a 379-bp cDNA template was amplified, and the PCR product was detected using SYBR® Green I chemistry. The experiment was repeated on a subsequent day. The experimental design was such that the data lent itself to analysis using an appropriate method for testing repeatability. It was found that, within a single assay, for samples assayed in triplicate, a difference of 23% may be reliably detected. Furthermore, when all of the factors that contribute to variability in the assay are taken into account, such as day-to-day variation in pipetting and amplification efficiency, a 52% difference in target template can be detected using a sample size of 4. The assay was found to be linear over at least four orders of magnitude.
Tissue-specific expression pattern of bovine prion gene:  Quantification using real-time RT-PCR
Ales Tichopad, Michael W. Pfaffl, Andrea Didier (2003)

Mol Cell Probes  17: 5-10

In recent studies PrP mRNA was determined mostly by in situ hybridisation or Northern Blot analysis—methods not suitable for absolute quantification of mRNA copy numbers. Herein we report on bovine prion mRNA quantification using calibrated highly sensitive externally standardized real-time RT-PCR with LightCycler instrument. Total RNA was isolated from nine different regions of the CNS and seven peripheral organs. PrPc mRNA copy numbers could be determined in all tissues under study. In approval with prior studies high mRNA level was found in Neocortex and Cerebellum. Lymphatic organs showed at least as high expression levels of prion mRNA as overall brain. Lowest expression was detected in kidney. Results of our study provide insight into the involvement of different organs in pathogenesis with respect to prion mRNA expression. LightCycler technology is currently considered the most precise method for nucleic acid quantification and showed to be powerful tool for further studies on prion diseases pathogenesis.

Figure: Mean PrPc mRNA copy numbers in various bovine tissue on mg tissue basis. Error lines indicate SD.


Mathematics of quantitative kinetic PCR and the application of standard curves
R. G. Rutledge* and C. Cote

Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du P.E.P.S., PO Box
3800, Sainte-Foy, Quebec G1V 4C7, Canada
Nucleic Acids Research, 2003, Vol. 31, No. 16 e93


Fluorescent monitoring of DNA ampliÆcation is the basis of real-time PCR, from which target DNA concentration can be determined from the fractional cycle at which a threshold amount of amplicon DNA is produced. Absolute quantification can be achieved using a standard curve constructed by amplifying known amounts of target DNA. In this study, the mathematics of quantitative PCR are examined in detail, from which several fundamental aspects of the threshold method and the application of standard curves are illustrated. The construction of Æve replicate standard curves for two pairs of nested primers was used to examine the reproducibility and degree of quantitative variation using SYBER‚ Green I fuorescence. Based upon this analysis the application of a single, wellconstructed standard curve could provide an estimated precision of 66±21%, depending on the number of cycles required to reach threshold. A simpliÆed method for absolute quantiÆcation is also proposed, in which quantitative scale is determined by DNA mass at threshold.

Instant evaluation of the absolute initial number of cDNA copies from a single real-time PCR curve
Stephane Swillens*, Jean-Christophe Goffard, Yoann Marechal,
Alban de Kerchove d'Exaerde1 and Hakim El Housni2
Nucleic Acids Research, 2004, Vol. 32, No. 6 e53
Amplification of a cDNA product by quantitative PCR (qPCR) is monitored by a fluorescent signalproportional to the amount of produced amplicon. The qPCR amplification curve usually displays anexponential phase followed by a non-exponential phase, ending with a plateau. Contrary to prevalentinterpretation, we demonstrate that under standard qPCR conditions, the plateau can be explained bydepletion of the probe through Taq polymerasecatalysed hydrolysis. Knowing the probe concentrationand the fluorescence measured at the plateau, a specific fluorescence can thus be calculated. As faras probe hydrolysis quantitatively reflects amplicon synthesis, this, in turn, makes it possible to convertmeasured fluorescence levels in the exponential phase into concentrations of produced amplicon. Itfollows that the absolute target cDNA concentration initially engaged in the qPCR can be directly estimatedfrom the fluorescence data, with no need to refer to any calibration with known concentrations of target DNA.
A method for the rapid construction of cRNA standard curves  in quantitative real-time RT-PCR.

Fronhoffs S, Totzke G, Stier S, Wernert N, Rothe M, Bruning T, Koch B,
Sachinidis A, Vetter H, Ko Y.
Mol Cell Probes  2002 Apr;16(2): 99-110

Medizinische Universitats-Poliklinik Bonn, Institut fur experimentelle
Dermatologie, Wilhelmstr. 35-37, Bonn, 53111 Bonn, Germany

Quantification of nucleic acids, especially of mRNA, is increasingly important in biomedical research. The recently developed quantitative real-time polymerase chain reaction (PCR) - a highly sensitive technology for the rapid, accurate and reproducible quantification of gene expression - offers major advantages over conventional quantitative PCR. Transcript quantification is performed in the exponential phase of the PCR reaction through extrapolation of fluorescence signals from a standard calibration curve which represents the initial copy number for a given fluorescence signal. We have developed a method for gene transcript quantification which is based on a LightCyclertrade mark - assisted real-time PCR in combination with a simple and rapid approach for the construction of external cRNA standards with identical gene sequences as the target gene. Synthesis of cRNAs was performed by in vitro transcription with T7 RNA polymerase followed by reverse transcription and real-time PCR. We applied this approach for transcript quantification of eukaryotic initiation factor 3 p110 (EIF3S8) mRNA in normal testicular tissue. We also present a rapid and simple strategy for the construction of cRNA standards for use in real-time PCR.

 
Tissue specific expression pattern of estrogen receptors (ER):
Quantification of ERa and ERb mRNA with real-time RT-PCR

Pfaffl MW, Lange IG, Daxenberger A, Meyer HH. 
                Tissue-specific expression pattern of estrogen receptors (ER): 
quantification of ER alpha and ER beta mRNA with real-time RT-PCR. 
APMIS. 2001 109(5): 345-55.

Summary

We have examined the tissue specific mRNA expression of ERa and ERb in various bovine tissues using real-time RT-PCR. Goal of this study was to evaluate the deviating tissue sensitivities and the influence of the estrogenic active preparation RALGRO on the tissue specific expression and regulation of both ER subtypes. RALGRO contains Zeranol (a-Zearalanol), a derivative of the mycotoxin Zearalenon, shows strong estrogenic and anabolic effects, and exhibits all symptoms of hyper-estrogenism in particular reproductive and developmental disorders. Eight heifers were treated over 8 weeks with multiple dose implantations (0x, 1x, 3x, 10x) of Zeranol. Plasma Zeranol concentration, measured by enzyme-immuno-assay, of multiple treated heifers Zeranol were elevated. To quantify ERa and ERb transcripts also in low abundant tissues, sensitive and reliable real-time RT-PCR quantification methods were developed and validated on the LightCycler. Expression results indicate the existence of both ER subtypes in all 15 investigated tissues. All tissue exhibit a specific ERalpha and ERbeta expression pattern and regulation. With increasing Zeranol concentrations a significant down-regulation of ERa mRNA expression could be observed in jejunum (p<0.001) and kidney medulla (p<0.05). These data support the hypothesis, that the ERb may have different biological functions than ERa, especially in kidney and the jejunum.

Introduction

Cloning and sequencing of ERb in human (1), rat (2), and mouse (3) has provided the first example of a steroid hormone receptor existing in two isoforms, each of which is encoded by a separate gene. The ERb protein is smaller than the previously identified ERa (4,5) but possesses the modular structure of distinct functional domains (A-F) characteristic of the members of the nuclear receptors. The DNA-binding domain of ERa and ERb is highly conserved over several species (>95% homology in Homo sapiens, Rattus norwegicus, Mus musculus, Bos taurus) and the ligand binding domain shows ~60% conserved residues (6-8). 

ERbeta seems to be an additional important factor in the mechanism of estrogen action and has an overlapping but non identical tissue distribution in comparison to ERa. It is expressed in humans and rodents in the central nervous system, the cardiovascular system, the immune system, and in the gastrointestinal tract (9-11). Within the same organ it often appears that the ER subtypes are expressed differentially, supporting the hypothesis that both receptors may have different biological functions. Ligand binding experiments have shown specific binding of estradiol-17b and transactivation of ERb with an affinity similar to ERa (12). But the mechanism of activation by endogen steroids, phyto-etrogens, xenosteroids and related synthetic drugs was shown to be very complex (6,13,14). Knowledge of the distribution and regulation of ERb in various tissues of ruminants are missing at this time. The available publications about ERb expression in bovids are limited to the cattle reproductive organs (8,15) and to the sheep hypothalamus (16,17). However, a more detailed study of the tissue distribution of both ER subtypes is essential to continue investigations of their regulation and physiological function. It is well known that steroids lead to an increased synthesis of specific proteins (18) and it is proposed that estradiol can stimulate via ERa its own receptor expression at least in the uterus (19). Goal of this study was to evaluate the deviating tissue sensitivities and the influence of the estrogen active preparation RALGRO ® (Mallinckrodt Veterinary, Inc., Mundelein, IL, USA) on the tissue specific expression pattern and regulation of both ER subtypes. One RALGRO implant contains 36 mg a-Zearalanol (Zeranol), a derivative of the mycotoxin Zearalenon, and shows strong estrogenic and anabolic effects in farm animals. Besides this, it exhibits all symptoms of hyper-estrogenism in particular reproductive and developmental disorders. Herein we describe a reverse transcription followed by a real-time polymerase-chain-reaction (RT-PCR) on the LightCycler system (Roche, Basel, Suisse) to detect and quantify these mRNA transcripts from both genes. For low abundancies, sensitive and reliable real-time RT-PCR quantification methods were developed and validated. RT-PCR real-time assays with an external calibration curve are quantitative and therefore an absolute comparison of ERa and ERb within one tissue RNA preparation will be possible. 



Conclusion

In view of the data provided for sensitivity, linearity and reproducibility, the developed RT-PCR assay developed herein allows the absolute and accurate quantification of ERa and ERb mRNA molecules with a sufficiently high sensitivity even for tissues with low abundancies down to a few molecules. Our expression results indicate the existence of two ER subtypes in various bovine tissues, their different expression pattern and co-expression as well as their tissue specific regulation under estrogen treatment. These different expression pattern of ERa and ERb can be regarded as support for the hypothesis that the ER subtype proteins may have different biological functions, especially in kidney and the jejunum where ERb expression ratio is inverse in comparison to the other investigated tissues. In future more detailed study of ERa and ERb must be investigated in all kidney cell types and all parts of the gastrointestinal system to continue investigations of the ER regulation and its physiological function.
Real-time RT-PCR quantification of insulin-like growth factor  (IGF)-1, IGF-1 receptor, IGF-2, IGF-2 receptor,
insulin receptor,  growth hormone receptor, IGF-binding proteins 1, 2 and 3 in the bovine species.

M.W. Pfaffl , T. Mircheva Georgieva, I. Penchev Georgiev, E. Ontsouka, M. Hageleit & J. W. Blum (2002)
 Domest Anim Endocrinol, 22(2): 91-102.

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Summary

Reverse transcription (RT) followed by polymerase chain reaction (PCR) is the technique of choice for analyzing mRNA in extremely low abundance. Real-time RT-PCR using SYBR Green I detection combines the ease and necessary exactness to be able to produce reliable as well as rapid results. To obtain highly accurate and reliable results in a real-time RT-PCR a highly defined calibration curve is needed. We designed and developed nine different calibration curves, based on recombinant DNA plasmid standards and established them on a constant real-time PCR platform for the following factors: growth hormone receptor (GHR), insulin-like growth factor (IGF)-1, IGF-1 receptor (IGF-1R), IGF-2, IGF-2 receptor (IGF-2R), insulin receptor (INSR), and IGF-binding proteins (IGF-BP) 1, 2 and 3. Developed assays were applied in the LightCycler system on bovine ileum and liver total RNA and showed high specifity and sensitivity of quantification. All assays had a detection  limit of under 35 recombinant DNA molecules present in the capillary. The SYBR Green I determination resulted in a reliable and accurate quantification with high test linearity (Pearson correlation coefficient r > 0.99) over seven orders of magnitude from <102 to >108 recombinant DNA start molecules and an assay variation of maximal 5.3%. Applicability of the  method was shown by analyzing mRNA levels in newborn calves: mRNA concentrations  per gram tissue of mRNAs of IGF-1, IGF-1R, IGF-2, IGF-2R, GHR, INSR, and IGFBP-1, -2 and -3 were all different between in liver and ileum and the traits all exhibited individual differences.

Discussion
In this study we have designed, optimised and validated nine assays of factors involved in the action of the somatotropic axis in real-time RT-PCR using the SYBR Green I technology with the LightCycler. The amplification of the PCR products
was shown to be linear over a wide range of input copies, with high sensitivity, precision and reproducibility. As few as 35 molecules could be detected with the established quantification models. Amplification of genomic DNA was avoided by primer pairs located on different exons, therefore a DNAse treatment of tissue total RNA samples was unnecessary. A great simplification for the determination at the mRNA level of the nine parameters involved in the somatotropic axis was achieved by use of only one cDNA for the determination of all parameters. This was done by a reverse transcription of total RNA extracted from the tissues using random hexamer primers. High reproducibility and low test variability of £ 5.3% could be derived. To characterize the RT-PCR variation at its best, an average variation coefficient was calculated over the whole range of the calibration curve. This reflects the realistic PCR variation over the complete 
quantification range [17]. An externally recDNA calibration curve mimics the real-time PCR better than other standard materials, like recombinant RNA or purified PCR products, and possesses an almost similar amplification efficiency as the native sample mRNA [4]. Double-stranded recDNA, derived from a linearized plasmid, is a stable and reliable standard material for calibration curves and will be not degraded over a long storage period [4]. Therefore the test variability is minimized, the repeatability of is maximized, and the derived expression results are fully comparable over all applied quantification tests. 
Table: PCR efficiency calculated according to the equation: E = 10 [–1/slope] [18]. The detection limit, quantification range and test linearity (r = Pearson correlation coefficient) are given in molecules per capillary. Assay variation was determined in three repeats (n = 3) over the complete quantification range. Determination of variation is based on crossing point variation and was done in 25 ng reverse transcribed total RNA.


 

Figure 1: High resolution 4% Agarose gel electrophoresis of all real-time RT-PCR products derived from bovine liver total RNA. Lane 1+13: length standard (2 kbp, 1.2 kbp, 800 bp, 400 bp, 200 bp, 100bp); Lane 3-11:   IGF-1 @ 240 bp; IGF-1R @ 314 bp; IGF-2 @ 205 bp; IGF-2R @ 144 bp; IGF-BP1 @ 123bp; IGF-BP2 @ 136 bp; IGF-BP3 @ 194 bp; GHR @ 138 bp; INSR @ 163 bp.


 

 All factors could be quantified with the new established assays with high exactness and reproducibility. The quantified mRNA concentrations and expression levels are different between liver and ileum. It turned out, that IGF-2R is very low abundant in both tissues, whereas expressions of GHR, IGFBP-1 and IGFBP-2 are very low in ileum, but are medium or even highly expressed in liver. Extremely high mRNA concentrations could be measured for IGF-2 and IGFBP-1 in liver. Except for IGF and IGF-1R all factors showed significant different expression levels in ileum versus liver – either on 25 ng cDNA basis (raw and normalized data) or on mg tissue basis. For IGF and IGF-1R only significant expression levels could be determined on mg tissue basis (P<0.05) and fg/pg mRNA/mg tissue (P<0.05). Ligand concentration in comparison to their corresponding receptor in the IGF-1 and IGF-2 systems showed extreme differences between the tissues. Ligand mRNAs were always higher expressed than receptors and showed ratios of ligand to receptor (IGF-1/IGF-1R) in liver (ratio = ~12) higher than in ileum (ratio = ~2.5). The IGF-2/IGF-2R ratio was more evident in both tissues and extremer in ileum (ratio = ~900) than in liver (ratio = ~125). Studies on the physiological relevance of these data with more tissues and under different physiological conditions are in progress. 

The developed assays containing recDNA calibration curve, specific primers and cycling conditions can be applied as well on other real-time quantification systems: TaqMan ® (PE Applied Biosystems, Foster City, CA, USA), RotoGene ® (Corbett Research, Sydney, NSW, Australia), iCycler ® Thermal Cycler (Bio-Rad, Hercules, CA, USA) and Multiplex Quantitative PCR System ® (Stratagene, La Jolla, CA, USA). But the assay performances described herein are optimised to the demands of the LightCycler platform. For the above mentioned real-time PCR machines assay performances like sensitivity, linearity, reproducibility and PCR efficiency must be separately validated for each used platform. Performance variation can occur on the basis of different cycling techniques, fast-cycle vs. conventional block-cycle technology, other fluorescence excitation techniques, laser vs. light emitting diode excitation, and the applied fluorescence detection system, CCD camera (charge coupled device image sensor) vs. photo-hybrid detection.

Conclusion

In conclusion, the sensitivity, linearity and reproducibility of the developed real-time PCR assays allows absolute and accurate quantification, down to a few molecules. In future we will use the established quantification systems to compare the expression rates in tissues of Bos taurus and other species like Homo sapiens, Ovis aries, Sus scrofa, Rattus norvegicus, and Callithrix jacchus to investigate physiological changes in gene expression.
Effects of synthetic progestagens on the mRNA Expression of 
Androgen Receptor, Progesterone Receptor, Estrogen Receptor ERa and ERb, Insulin-like Growth Factor (IGF)-1
and IGF-1 receptor in Heifer tissues

M.W. Pfaffl, A. Daxenberger, M. Hageleit & H.H.D. Meyer (2002)
J Vet Med A 49: 57-64

SUMMARY

Synthetic progestagen like melengestrol acetate (MGA) are widely used for estrus synchronisation and for growth promotion in cattle production. The metabolic effects exceed its primary potency as a progestagen. It is speculated that MGA stimulates follicle development and thereby endogenous estrogen production, but inhibits ovulation. To investigate the dose dependent effects on the mRNA expression levels, six heifers were fed during 8 weeks with different levels of MGA (0.5 mg, 1.5 mg, 5 mg) daily and two heifers served as control. The expression of steroid receptor mRNA [androgen receptor (AR), progesterone receptor (PR), estrogen receptor (ER) ERa and ERb], insulin-like growth factor-1 (IGF-1) and its receptor were quantified in liver, neck (m. splenius) and shoulder muscularity (m. deltoideus). Plasma concentrations of IGF-1 were quantified by radioimmunoassay. In treated animals the MGA plasma levels were elevated over the complete treatment period, corresponding to the MGA treatment concentrations. IGF-1 concentrations of control animals were at constant levels. Plasma levels for estradiol (E2) and IGF-1 were increased in low MGA treatment group. Overdosed MGA decreased progesterone (P4) and E2 levels. To quantify the IGF-1 and all receptor mRNA transcripts, sensitive and reliable real-time RT-PCR quantification methods were developed and validated in the LightCycler. A dose dependent relationship between increasing MGA concentrations and mRNA expression were observed in liver for AR and IGF-1 receptor, and in neck muscularity for IGF-1. ERa in liver and neck muscle showed a trend of increasing expression.

CONCLUSION

Expression results indicate the existence of AR, ERa, ERb, IGF-1 and IGF-1 receptor in various bovine tissues, their different expression pattern and regulation under progestagen treatment. In view of the data provided for sensitivity, linearity and reproducibility, the developed RT-PCR assay developed herein allows the absolute and accurate quantification of mRNA molecules with a sufficiently high sensitivity. Presented results as well as results derived from Daxenberger et al. (1999) and Hageleit et al. (2000) can be concluded as summarized in table 6. MGA could stimulate the follicle and thereby inhibit ovulation. An effect on the plasma hormone profiles and anabolism was evident. Plasma concentration showed elevated MGA levels in treated animals, and therefore significant decreased levels of P4 and E2. IGF-1 concentrations seemed only elevated in the low treatment group. Still we found positive correlations between MGA concentrations and mRNA expression in liver for AR and IGF-1 receptor, as well as in neck muscularity for IGF-1. ERa showed a trend of increasing expression. For PR no quantitative RT-PCR product could be generated in the investigated tissues. Thus other mechanisms different from the direct steroid-receptor mechanism are likely to regulate IGF-1 and ERb expression.

Table:  Synopsis of the knowledge about the anabolic mechanism of MGA action

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