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


The gastrointestinal tract as target of steroid hormone action:
Quantification of steroid receptor mRNA expression (AR, ER-alpha, ER-beta and
PR) in 10 bovine gastrointestinal tract compartments by kinetic RT-PCR

M.W. Pfaffl, I.G. Lange, H.H.D. Meyer
Journal of Steroid Biochemistry & Molecular Biology (2003) 84 (2-3): 159-166

SUMMARY
We have examined the tissue-specific mRNA expression pattern of androgen receptor (AR), both estrogen receptor (ER) subtypes ER-alpha and ER-beta and progestin receptor (PR) in 10 bovine gastrointestinal compartments. Goal of this study was to evaluate the deviating tissue sensitivities and the influence of the estrogenic active preparation Ralgro® on the compartment-specific expression regulation. Ralgro® contains Zeranol which shows strong estrogenic and anabolic effects. Eight heifers were treated for 8 weeks with Ralgro® at different dosages (0, 1, 3, and 10 times). To quantify the very lowabundant steroid eceptormRNAtranscripts sensitive and reliable real-time (kinetic) reverse transcription (RT)-PCR quantification methods were validated on the LightCycler. Expression results indicate the existence of AR and both ER subtypes in all 10 gastrointestinal compartments. PR receptor was expressed at very low abundancy. Gastrointestinal tissues exhibit a specific ER-alpha and ER-beta expression pattern with high expression levels for both subtypes in rectum, colon and ileum.With increasing Zeranol concentrations a significant down-regulation for ER-alpha and ER-beta was observed in jejunum (P < 0 .001 and P<0.05, respectively). Significant up-regulations under estrogen treatment could be shown in abomasum for ER-alpha (P < 0.05) and in rectum for ER-beta (P < 0.001). The authors conclude, that especially estrogens and the expression of their corresponding receptor subtypes may play an important role in the modulation and regulation in gastric as well as gut functions, cell proliferation and possibly in the pathophysiology of cell cancer. The different expression patterns of ER-alpha and ER-beta can be regarded as support of the hypothesis that the subtype proteins may have different biological functions in the gastrointestinal tract. AR and PR seem to be not estrogen dependent.


Expression and localisation of oestrogen and progesterone receptors in the
bovine mammary gland during development, function and involution.

Schams D, Kohlenberg S, Amselgruber W, Berisha B, Pfaffl MW, Sinowatz F.
J Endocrinol  2003 May;177(2): 305-317
Institute of Physiology, Technical University Munich-Weihenstephan, D-85350, Freising, Germany.
It is now well established that oestrogen and progesterone are absolutely essential for mammary gland development.
Lactation can be induced in non-pregnant animals by sex steroid hormone treatment. Most of the genomic actions
of oestrogens are mediated by two oestrogen receptors (ER)-alpha and ERbeta, and for gestagens in ruminants
by the progesterone receptor (PR). Our aim was the evaluation of mRNA expression and protein (localisation and
Western blotting) during mammogenesis, lactogenesis, galactopoiesis (early, middle and late) and involution
(8, 24, 28, 96-108 h and 14-28 days after the end of  milking) in the bovine mammary gland (total no. 53).
During these stages, the mRNA was assessed by means of real-time RT-PCR (LightCycler). The protein for
ERalpha, ERbeta and PR was localised by immunohistochemistry and Western blotting. The mRNA expression
results indicated the existence of ERalpha, ERbeta and PR in bovine mammary gland. Both ERalpha and
PR are expressed in fg/ micro g total RNA range. The highest mRNA expression was found for ERalpha and
PR in the tIssue of non-pregnant heifers, followed by a significant decrease to a lower level at the time
of lactogenesis with low concentrations remaining during lactation and the first 4 weeks of involution.
In contrast, the expression of ERbeta was about 1000-fold lower (ag/ micro g total RNA) and showed no
clear difference during the stages examined, with a significant increase only 2-4 weeks after the end of milking.
Immunolocalisation for ERalpha revealed a strong positive staining in nuclei of lactocytes in non-pregnant
heifers, became undetectable during pregnancy, lactogenesis and lactation, and was again detectable 14-28
days after the end of milking. In contrast, PR was localised in the nuclei of epithelial cells in the mammary tIssue
of non-pregnant heifers, in primigravid animals, and during late lactation and involution. During lactogenesis,
peak and mid lactation, fewer nuclei of epithelial cells were positive, but increased staining of the cytoplasm of
epithelial cells was obvious. ERalpha and ERbeta protein was found in all mammary gland stages
examined by Western blotting. In contrast to mRNA expression, the protein signal for ERalpha was
weaker in the tIssue of non-pregnant heifers and during involution (4 weeks). ERbeta protein showed a stronger signal
(two isoform bands) in non-pregnant heifers and 4 weeks after the end of milking. This correlated with the mRNA expression
data. Three isoforms of PR (A, B and C) were found by Western blotting in the tIssue of non-pregnant heifers, but only
isoform B remained during the following stages (lactogenesis, galactopoiesis and involution). In conclusion, the mRNA
expression and protein data for ER and PR showed clear regulatory changes, suggesting involvement
of these receptors in bovine mammary gland development and involution.


Expression of estrogen and progesterone receptors in the bovine ovary during estrous cycle and pregnancy.

Berisha B,  Pfaffl MW,  Schams D.
Endocrine  2002 Apr;17(3):207-214

The objective of the study was to demonstrate the mRNA expression of estrogen receptor a (ERa), ERbeta, and progesterone receptor (PR) by block reverse transcription-polymerase chain reaction(RT-PCR) and real-time RT-PCR (LightCycler) in bovine ovarian follicles and in corpus luteum during the estrous cycle and pregnancy. The mRNA expression of ERalpha and ERbeta mRNA in theca interna tissue (TI) (lower pg/microg RNA) increased continuously and significantly during final growth of follicles, with much higher levels for ERalpha. The mRNA expression of ERalpha and ERbeta in granulosa cells (GC) (fg/microg RNA) increased continuously during follicle growth but without any significant change. The expression of mRNA for PR in follicles (lower fg/microg RNA) increased continuously to maximum level in preovulatory follicles with a significant change only in TI. The highest mRNA expression for ERalpha (fg/microg RNA) was detected in corpus luteum (CL) during the early luteal phase, following by a significant decrease of expression during the mid, late,
and regression phases. In contrast, ERbeta mRNA expression is relatively high during the early stage, decreased during the late early and mid luteal phase, and increased significantly again during the late luteal phase and after CL regression. During pregnancy (>3 mo), low levels of ERalpha and ERbeta mRNA expression (<25 fg/microg RNA) with no significant changes were measured. No significant change in PR mRNA expression (levels <13 fg/microg RNA) during the estrous cycle and pregnancy in bovine CL were found. The results suggest an autocrine/paracrine role of steroid receptors in the regulation of final follicle growth and corpus luteum formation and function.

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 May;109(5): 345-55.

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.

Effects of synthetic progestagens on the mRNA Expression of Androgen Receptor, Progesterone Receptor, Estrogen Receptor 
ER-alpha and ER-beta, 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 (2992) 49: 57-64

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.

Effects of muscle type, castration, age, and compensatory growth rate on androgen receptor mRNA expression in bovine skeletal muscle

Brandstetter AM, Pfaffl MW, Hocquette JF, Gerrard DE, Picard B, Geay Y, Sauerwein H. ( 2000)
 J Anim Sci. 2000 Mar;78(3):629-37.

The effect of testosterone on sexual dimorphism is evident by differential growth of forelimb and neck muscles in bulls and steers. Divergent hormone sensitivites may account for the differential growth rates of individual muscles. Therefore, the objective of this study was to compare androgen receptor (AR) expression in three different muscles of bulls and steers at various ages and growth rates. Thirty Montbeliard bulls and 30 steers were assigned to four slaughter age groups. Four or five animals of each sex were slaughtered at 4 and 8 mo of age. Animals in the remaining two slaughter groups (12 and 16 mo) were divided into groups of either restricted (R) or ad libitum (AL) access to feed. Five animals of each sex and diet were slaughtered at the end of the restricted intake period at 12 mo of age. To simulate compensatory growth, the remaining animals (R and AL) were allowed ad libitum access to feed until slaughter at 16
mo of age. Total RNA was extracted from samples of semitendinosus (ST), triceps brachii (TB), and splenius (SP) muscles. Androgen receptor mRNA was quantified in 200-ng total RNA preparations using an internally standardized reverse transcription (RT) PCR assay. Data were analyzed using 18S ribosomal RNA concentrations as a covariable. Steers had higher AR mRNA levels per RNA unit
than bulls (P < .01). Androgen receptor mRNA levels differed between muscles (P < 0.05), with lowest expression in the SP. The pattern of AR expression differed (P < 0.05) for each muscle with increasing age. Between 4 and 12 mo of age, AR mRNA levels increased (P <  0.05) in SP but remained unchanged in the ST and TB. Feeding regimen had no effect on muscle AR expression, but steers exhibiting compensatory growth had higher AR mRNA levels than AL steers (P < 0 .01) or bulls (P < 0 .01). Our results show that AR expression is muscle-specific and may be modulated by circulating testicular hormones. These data suggest that the regulation of AR expression may be linked to allometric muscle growth patterns in cattle and compensatory gain in steers.

Quantification of androgen receptor mRNA in tissues by competitive co-amplification of a template in RT-PCR

Malucelli A, Sauerwein H, Pfaffl MW, Meyer HH.  (1996)
J Steroid Biochem Mol Biol. (1996) 58(5-6): 563-568

We describe a polymerase chain reaction (PCR)-based method for the quantification of androgen receptor (AR) mRNA in tissues. The amount of PCR products depends on the exponential amplification of the initial cDNA copy number; therefore minor differences in the efficiency of amplification may dramatically influence the final product yield. To overcome these tube-to-tube differences in reaction efficiency, an internal control AR cRNA was reverse transcribed along with the target mRNA using the same primers. This standard was
obtained by deleting a 38 bp fragment from an amplified bovine AR sequence, which was then subcloned and transcribed into cRNA. Known dilutions of the competitor cRNA were spiked into a series of RT-PCR reaction tubes containing equal amounts of the target mRNA. Following RT-PCR, the co-amplified specimens obtained were separated by gel electrophoresis and quantified by densitometric
analysis of ethidium bromide stain. We applied this method to quantify the AR-mRNA in skeletal muscle of castrated as well as from intact male cattle. The applicability of the quantification system for AR-mRNA described herein was demonstrated for other species, e.g. man.

Expression of estrogen and androgen receptor in the bovine gastrointestinal tract

Sauerwein H.; Pfaffl M.; Hagen-Mann K.; Malucelli A. & Meyer H.H.D. (1995)
Deutsche tierärztliche Wochenschrift 1995, 102: 164-168.

Reproductive and maturational nutritive needs are examples for situations in which alterations in circulating concentrations of sex steroids are associated with changes in gastrointestinal function. In order to investigate whether there is a causal relationship between sex steroids and gastrointestinal function, we aimed to investigate the responsiveness for androgens and for estrogens of the bovine gastrointestinal tract. Using Northern blot analysis, estrogen receptor (ER) mRNA was detected in rumen tissue. Comparing the ER expression in rumen from females of different reproductive stages, we found that no differences related to cycle stage, pregnancy or parturition could be detected. In contrast, the ER expression rates in the uterus of the respective animals showed the same dependency of reproductive stage as demonstrated earlier for the ER protein, indicating that there might be a tissue specific regulation of ER. By in-situ hybridization of rumen tissue sections the expression of ER was localized in the epithelium of the papillae. In the muscular layer no positive signals for ER mRNA were observed. Above rumen, the presence of ER and androgen receptor (AR)
mRNA was determined in various intestinal tissues using reverse transcription (RT) and polymerase chain reaction (PCR). Primers were selected from the bovine androgen and estrogen receptor sequence to amplify parts of the sequence coding for the hormone binding part of the respective receptor. The PCR amplifies were subsequently electrophoresed on 1% agarose gels and visualized by ethidium
bromide staining. ER mRNA expression was demonstrated in reticulum, omasum, abomasum, duodenum, jejunum, ileum, caecum and colon. AR mRNA expression was not determined in the forestomaches, but was present in all intestinal segments investigated.

Uterine Androgen Receptor mRNA Expression in Metestrous and 
Anestrous Bitches being healthy or suffering from pyometra

SauerweinH., A. Brandstetter, M.W. Pfaffl, H.H.D. Meyer, E. Möstl, J. Handler & K. Arbeiter (1998)
Deutsche tierärztliche Wochenschrift 105: 173-208.

Summary
The importance of androgens for the female reproductive system has been investigated for decades and a number of androgen sensitive processes has now been identified in female reproductive organs. For carnivore species no data were available so far about uterine androgen sensitivity and its regulation. The present study therefore aimed to investigate whether androgen receptors (AR) are present in the dog uterus, whether they are regulated throughout the ovarian cycle and whether pyometra affects their expression rate. Uterine tissue samples were collected from 28 bitches of different ages and various breeds. The samples were grouped according to the stage of estrous cycle (metestrus ME or anestrus AE) and the pathological status of the uterus (i.e. suffering from pyometra or not). Androgen receptor mRNA (AR mRNA) was quantified from 500 ng of total RNA isolated from the tissue samples using an internally standardized reverse transcription polymerase chain reaction (RT-PCR) described previously. The amount of total RNA extractable per g tissue was elevated during pyometra. The successful amplification of the expected 172 bp fragment from canine uterine RNA
together with the confirmation of the identity of this fragment by sequence analysis, demonstrates that AR is expressed in this particular tissue. Comparing the expression rates in uteri from bitches during ME or AE being healthy (H) or suffering from pyometra (P), the only significant (p < 0.01) difference was found between H and P uteri during ME with 3.5-fold lower expression rates in P.
Although the same seems true for AE bitches, a significant difference could not be demonstrated due to the low number (n = 2) of diseased animals in the AE group. There was no evident effect of the stage of ovarian cycle on uterine AR mRNA levels.
Characterisation of gene expression patterns in 22RV1 cells for
determination of environmental androgenic/antiandrogenic compounds

A. Hartel, A. Didier, M.W. Pfaffl, Heinrich H.D. Meyer
Journal of Steroid Biochemistry & Molecular Biology (2003) 84 (2-3):  231-238

SUMMARY
Alteration of androgen receptor function due to hormonally active compounds in the environment, may be responsible for impaired reproductive function in aquatic wildlife. Based on human prostate carcinoma 22RV1 cells, a cell culture expression system was established to test effects of putative androgenic/antiandrogenic compounds on endogenous gene expression. 22RV1 cells were shown to express human androgen receptor, but not human progestin (hPR) or human oestrogen receptor (hER) alpha and beta. Six androgen-regulated genes (ARGs) were chosen to determine androgenic/antiandrogenic action using highly sensitive real-time RT-PCR. Results showed that gene expression is altered in a time-dependent manner. After stimulation of cells by DHT (10 nM), synthetic androgen R1881 (1 nM), or organic pesticides (difenoconazole, fentinacetate, etramethrin) TMPRSS2 mRNA expressionwas down-regulated by the factor 0.6 after 24 h ofDHTtreatment. Similar results were obtained when cells were assayed for mRNA expression of PSA after fentinacetate and R1881 stimulation. In contrast, TMPRSS2 expression was up-regulated by the factor 0.9 when cells were stimulated by tetramethrin. Final goal of the work is a sensitive determination of differential gene expression by different compounds under study, achievement of substance-specific expression patterns and function related analysis of potential  androgens/antiandrogens.
Detection and quantification of mRNA-expression of alpha- and beta-adrenergic
receptor subtypes  in the mammary gland of dairy cows

T. Inderwies, M. W. Pfaffl, H. H. D. Meyer, J. W. Blum & R. M. Bruckmaier
Domestic Animal Endocrinology (24): 123-135

Summary
Adrenergic receptors are pharmacologically classified into the receptor types alpha-1, alpha-2, beta-1, beta-2, and beta-3. Structural differences and varying affinities in radioligand binding studies lead to a further classification of alpha-1- and alpha-2-receptors into subtypes which are termed alpha-1A (formerly C), alpha-1B, and alpha-1D-(formerly AD), and alpha-2AD, alpha-2B, and alpha-2C, respectively. mRNA-expression of all but one alpha-adrenergic receptor subtypes and of all beta-adrenergic receptor types was measured quantitatively in total RNA extracted from mammary tissue of ten lactating dairy cows by real-time reverse transcriptase (RT) polymerase chain reaction (PCR). mRNA expression of alpha1-adrenergic receptors was highest for the alpha-1A-subtype followed by alpha-1B, whereas the alpha-1D-subtype could not be detected. The highest mRNA expression of alpha-2-adrenergic receptors was found for the alpha-2AD-subtype, followed by alpha-2B and alpha-2C. Within the beta-adrenergic receptors, the beta-2-receptor type was most highly expressed, followed by beta-1 and beta-3. In conclusion, eight of nine adrenergic receptors classified to date were detected and relatively quantified in the mammary gland of dairy cows.

New sequences of alpha-adrenergic receptor subtype mRNA 
at  GENBANK and EMBL

Milking characteristics and their relation to adrenergic receptor mRNA expression
and ligand binding in the mammary gland of dairy cows

T. Inderwies, M. W. Pfaffl & R. M. Bruckmaier
Domestic Animal Endocrinology 2003 (25): 275-286


Stimulation of small alpha, Greek - and small beta, Greek -adrenergic receptors in the bovine mammary gland affects milking characteristics such as milk yield and peak flow rate. The aim of this study was to detect possible correlations between milkability, adrenergic receptor binding capacity and receptor expression at the mRNA level. In addition, dose–response relationships of small alpha, Greek - and small beta, Greek -adrenergic receptor stimulation were evaluated after application of an small alpha, Greek - and small beta, Greek -adrenergic receptor agonist, respectively in different dosages. Density and distribution of adrenergic receptor binding sites in the region around the large mammary ducts were investigated as well as adrenergic receptor mRNA expression. Milk flow of one-quarter was recorded in 10 cows without or with additional small alpha, Greek - and small beta, Greek -adrenergic receptor stimulation in three dosages each. After slaughter, mammary tissue was taken from the region around the large mammary ducts in the previously investigated quarters. Protein and RNA were extracted for measuring small alpha, Greek 1-, small alpha, Greek 2-, and small beta, Greek 2-adrenergic receptor binding sites and mRNA expression levels by real-time polymerase chain reaction (RT-PCR). Peak flow rate without additional adrenergic receptor stimulation was negatively correlated with small alpha, Greek 2-adrenergic receptor binding (maximal binding capacity, Bmax) and positively correlated with small alpha, Greek 2-adrenergic receptor expression at the mRNA level (crossing point (CP) of the real-time PCR). During small alpha, Greek -adrenergic receptor stimulation, there was a positive correlation between milkability and small alpha, Greek 2-adrenergic receptor mRNA expression, whereas during small beta, Greek -adrenergic receptor stimulation no correlations were detected. Dose–response relationships were existing during small alpha, Greek -adrenergic receptor stimulations, but not during small beta, Greek -adrenergic receptor stimulations at four dosages each including control milking. Significant changes in milk yield and peak flow rate mainly occurred after application of an small alpha, Greek -adrenergic receptor agonist. In conclusion, high mRNA expression levels or binding capacities of adrenergic receptors do not necessarily lead to according reactions in vivo, concerning milk yield and peak flow rate. To influence milking characteristics, individual reactions of the cow on adrenergic stimulation have to be considered.
The mRNA expression of the members of the IGF-system
in bovine corpus luteum during induced luteolysis

T.P. Neuvians, M.W. Pfaffl, B. Berisha, D. Schams (2003)
Domest Anim Endocrinol, (25) 359-372


The components of the IGF-system were shown to be differentially regulated in bovine antral
follicles and corpora lutea (CL) during different stages of the estrous cycle, and to have impor-
tant functions for specific stages. The aim of this study was to investigate the detailed pattern
of mRNA expression of most constituents of the IGF-system and their possible involvement in
prostaglandin (PG)F2-alpha-induced luteolysis in the bovine CL. Therefore, cows in the mid-luteal
phase (days 8–12) were injected with the PGF2-analogue Cloprostenol, and CL were collected by
transvaginal ovariectomy at 2, 4, 12, 48 and 64 h after PGF2-injection. Real-time RT-PCR using
SYBR Green I detection was employed to determine mRNA expressions of the following factors:
ubiquitin (UBQ), insulin-like growth factor I (IGF I), IGF II, IGF-receptor type 1 (IGFR-1), growth
hormone receptor (GH-R) and IGF-binding proteins-1–6 (IGFBP-1–6). Total extractable RNA de-
creased with ongoing luteolysis. IGFBP-1 mRNA was significantly up-regulated at 2 h after PGF2-alpha
and maximal at 4 h with a 34-fold increase. IGFBP-5 mRNA was significantly up-regulated after
12 h with a maximum of an 11-fold increase at 64 h. For GH-R, IGFR-1, IGF II, IGFBP-3 and -4
mRNA expression, we found a significant down-regulation in certain stages. There was a significant
up-regulation for IGFBP-2 and -6 mRNA at 64 h after induced luteolysis. There were no significant
changes in IGF I mRNA expression. In conclusion, the IGF-system with all its components seems
to play an important role in the very complex process of PGF2-alpha-induced luteolysis in bovine CL.
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.

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.

Abundance of message for insulin-like growth factors-I and -II and for receptors for growth hormone, insulin-like growth factors-I and -II, and insulin in the intestine and liver of pre- and full-term calves

T. M. Georgieva, I. P. Georgiev, E. Ontsouka, H. M. Hammon, M. W. Pfaffl & J. W. Blum
Journal of Animal Science 81:  2294-2300

The somatotropic axis and insulin are involved in pre- and postnatal development. In pre- and full-term calves (GrPo and GrNo; born after 277 and 290 d of pregnancy, respectively) and in preterm calves on d 8 of life after being fed for 7 d (GrP8), we studied whether there are differences in the abundance of messenger RNA (mRNA) of IGF-I and IGF-II and of receptors for GH, IGF-I, IGF-II, and insulin among different intestinal sites (duodenum, jejunum, ileum, and colon) and whether there are ontogenetic differences during the perinatal period in intestine and liver. Intestinal site differences (P < 0.05) existed in mRNA levels of IGF-I and IGF-II and receptors for GH, IGF-I, IGF-II, and insulin. Abundance of mRNA of IGF-I and -II and of receptors for IGF-I and GH was highest (P < 0.05) in the colon, abundance of the receptor for IGF-II was comparably high in the colon and ileum, and that of the receptor for insulin was similarly high in colon, ileum, and jejunum. Among GrPo, GrNo, and GrP8 groups, there were differences (P < 0.05) in mRNA levels of IGF-I and IGF-II and of receptors for GH, IGF-I, IGF-II and insulin. Abundance of mRNA of IGF-I and IGF-II and of receptors for GH, IGF-I, IGF-II and insulin was highest (P < 0.05) in GrPo calves immediately after birth and was primarily seen in the ileum. In liver, the mRNA levels differed (P < 0.05) among groups for IGF-II and receptors for IGF-I, IGF-II, and insulin, and were highest (P < 0.05) for IGF-II in GrPo, for receptors of IGF-I in GrNo, and were higher (P < 0.05) in GrPo than GrP8 for receptors of IGF-II. In conclusion, mRNA levels of IGF-I and IGF-II and of receptors for GH, IGF-I, IGF-II, and insulin were different at different intestinal sites and in intestine and liver and changed during the perinatal period.

Effects of dexamethasone and colostrum intake on the somatotropic axis in neonatal calves
Sauter SN, Ontsouka E, Roffler B, Zbinden Y, Philipona C, Pfaffl MW, Breier BH, Blum JW, Hammon HM (2003)

Division of Animal Nutrition and Physiology, University of Berne, Institute of
Animal Genetics, Nutrition and Housing, Faculty of Veterinary Medicine, Berne,
Switzerland.

Am J Physiol Endocrinol Metab  285: E252-261 (2003) 

Glucocorticoids and colostrum feeding influence postnatal maturation of the somatotropic axis. We have tested the hypothesis that dexamethasone (DEXA) affects the somatotropic axis in neonatal calves dependent on colostrum intake. Calves were fed either with colostrum or with a milk-based formula (n = 14 per group) and in each feeding group, half of the calves were treated with DEXA (30 µg/[kg body weight × day]). Pre- and postprandial blood samples were taken on days 1, 2, 4, and 5 and liver samples were taken on day 5 of life. DEXA increased insulin-like growth factor (IGF)-I, but decreased growth hormone (GH) and IGF binding protein (IGFBP)-1 and -2 plasma concentrations and increased GH receptor (GHR) mRNA levels in liver. DEXA increased IGF-I mRNA levels only in formula-fed calves and increased hepatic GHR binding capacity, but only in colostrum-fed calves. Colostrum feeding decreased IGFBP-1 and -2 plasma concentrations and hepatic IGFBP-2 and -3 mRNA levels. In conclusion, DEXA and colostrum feeding promoted maturation of the somatotropic axis. DEXA effects partly depended on whether colostrum was fed or not.

Insulin-like growth factor and insulin receptors in intestinal mucosa of neonatal calves.
I P Georgiev, T M Georgieva, M Pfaffl, H M Hammon and J W Blum (2003)
Journal of Endocrinology, 176: 121-132

SUMMARY
Intestinal development is modified by age and nutrition, mediated in part by insulin-like growth factors (IGF-I, IGF-II) and insulin. We have investigated whether expression of IGF-I, IGF-II and insulin receptors (IGFIR, IGF-IIR and IR; measured by real-time RT-PCR) and binding capacity (Bmax) of IGF-IR, IGF-IIR and IR in the mucosa of the small and large intestine of neonatal calves are modified by age and di?erent feeding regimes. In experiment 1, pre-term (GrP) and full-term (GrN) calves (after 277 and 290 days of pregnancy respectively) were killed immediately after birth before being fed; a further group of full-term calves were fed for 7 days and killed on day 8 of life (GrC1–3). In experiment 2, full-term calves were killed on day 8 after being fed first-colostrum for 7 days (GrCmax), colostrum of the first  six milkings for 3 days (GrC1–3) or milk-based formula for 3 days (GrF1–3). Intestinal sites di?ered with respect to expression levels of IGF-IR (duodenum>jejunum in GrC1–3; ileum>colon, duodenumjejunum in GrF1–3), IGF-IIR (colon>duodenum and ileum in GrN), and IR (lowest in ileum in GrP and CrN; highest in colon in GrC1–3 and GrCmax). They also differed with respect to Bmax of IGF-IR (ileum and colon>duodenum and jejunum in GrP; ileum and colon>jejunum in GrN; colon>jejunum in GrC1–3; lowest in jejunum in GrF1–3), IGF-IIR (duodenum and colon>jejunum and ileum in GrP; duodenum>ilem and colon>jejunum in GrN; duodenum, jejunum and colon>ileum in GrCmax, GrC1–3, and GrF1–3) and IR (ileum>duodenum, jejunum and colon in GrCmax, GrC1–3, and GrF1–3). There were significant differences between groups in the expression of IGF-IR (GrF1–3> GrCmax and GrC1–3 in ileum), IGF-IIR (GrN>GrP and GrC1–3 in colon; GrN>GrC1–3 in jejunum and total intestine), and IR (GrCmax>GrF1–3 in colon) and in the Bmax of IGF-IR (GrP>GrN in colon; GrCmax>GrF1–3 in jejunum), IGF-IIR (GrN>GrP in duodenum, ileum and total intestine; GrN>GrC1–3 in duodenum, ileum, colon and total intestine) and IR (GrN>GrP in total intestine; GrC1–3>GrN in ileum and total intestine). In addition, Bmax values of IGF-IR, IGF-IIR and IR were correlated with villus circumference, villus height/crypt depth and proliferation rate of crypt cells at various intestinal sites. There were marked differences in Bmax of IGF-IR, IGF-IIR and IR dependent on mRNA levels, indicating
that differences in Bmax were the consequence of differences in posttranslational control and of receptor turnover rates. In conclusion IGF-IR, IGF-IIR and IR expressions and Bmax in intestinal mucosa were different at different intestinal sites and were variably affected by age, but not significantly affected by differences in nutrition. Receptor densities were selectively associated with intestinal mucosa growth.

Quantification of insulin-like growth factor-1 (IGF-1) mRNA: 
development and validation of an internally standardised competitive RT-PCR

Pfaffl M, Meyer HH, Sauerwein H.  (1998)
Exp Clin Endocrinol Diabetes. 1998;106(6):506-13.

Summary
To investigate the role of local IGF-1 mRNA expression in various tissues, we developed and validated a method which allows for a specific, sensitive and reliable quantification of IGF-1 mRNA: an internally standardised Reverse Transcription-Polymerase Chain Reaction (RT-PCR). A synthetic competitive template IGF-I standard cRNA (IGF-1 cRNA) was designed, which contains the same flanking primer sequences used to amplify the wild type IGF-1 mRNA, but differs by 56 bp in length. To obtain the IGF-1 mRNA concentration present in tissue RNA samples, series of 250 ng total-RNA were spiked with three known quantities of the standard IGF-1 cRNA, incubated for competitive RT-PCR reactions and the two amplificates obtained (184 bp from IGF-1 cRNA and 240 bp from the wild type IGF-I mRNA) were subsequently separated and quantified by HPLC-UV. For every individual tissue RNA sample, the ratio R (R = competitor PCR product / wild type PCR product) was plotted against the number of starting molecules of the competitor IGF-1 cRNA. The initial amount of IGF-1 mRNA present in the sample can then be read off where R = 1. The validated assay had a detection limit of 1600 IGF-1 cRNA molecules/reaction, the intra-assay variation was 7.4% (n = 5) and linearity (r = 0.997) was given between 140 ng to 840 ng total-RNA input. The present method was first applied to study the effect of long term castration on the IGF-1 expression rates in bovine tissues. The hepatic IGF-1 mRNA concentrations were well correlated (r = 0.81) with the plasma concentrations as quantified by RIA and were higher in intact than in castrated animals. In two skeletal muscles (m. splenius and m. gastrocnemius) IGF-1 mRNA concentrations were 20- and 35- times lower than in liver, respectively, without any differences between steers and bulls. In bulls, the IGF-1 mRNA expression was higher in m. splenius (p < 0.01) than m. gastrocnemius, indicating that locally produced IGF-1 might be important for sexually dimorphic muscle growth patterns.

Quantification of the insulin like growth factor-1 (IGF-1) mRNA:
Modulation of growth intensity by feeding results in inter- and 
intra-tissue specific differences of IGF-1 mRNA expression in steer

M. Pfaffl; F. Schwarz & H. Sauerwein (1998)s
Experimental and Clinical Endocrinology & Diabetes 106: 513-520.

Summary
The effect of constant and compensating body growth velocities on IGF-1 mRNA expression was studied in various tissues of growing steers. Twenty-six steers were allocated to three groups in which the average daily gains were kept either constantly high on intensive feeding, low on pasture feeding or were accelerated to compensatory growth after feed restriction. All animals were slaughtered at 570+/-2.6 kg and samples were collected from liver, heart, kidney and from 4 different muscles (m. splenius, m. soleus, m. cutaneus truncii and m. semispinalis capitis), which were selected in order to include maximal differences in fibre composition as well as in growth impetus. IGF-1 mRNA was quantified by a validated internally standardised RT-PCR method. The amount of RNA extracted from the various tissues investigated was constant within each type of tissue and showed no differences between treatment groups. As indicated by a constant ratio between the amount of RNA extracted and the DNA concentrations, there was no effect of the feeding on total transcriptional activity. The order of IGF-1 mRNA abundance per g tissue was liver > > kidney > heart > skeletal muscle. The different feeding regimen resulted in significant differences of IGF-1 mRNA expression rates in all organs showing different patterns between organs. IGF-1 mRNA concentrations showed muscle specific differences and also divergent reactions in response to the differing growth rates. These results support that the liver is the main IGF-1 producing tissue; above that they indicate that skeletal muscle, in particular when taking its absolute mass into account, might considerably contribute to the IGF-1 levels in blood. Our findings demonstrate that IGF-1 mRNA expression is regulated tissue specifically not only between different organs but also within musculature.

Effects of synthetic progestagens on the mRNA Expression of 
Androgen Receptor, Progesterone Receptor, Estrogen Receptor 
ER-alpha and ER-beta, 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.

Insulin-like growth factor-1 gene cloning and protein expression 
in bovine trabecular Meshwork tissue and cells

Yang Cao, Houren Wei, Da Banghong, Michael W. Pfaffl  & Li Zhongyu (2002)
 Department of Ophthalmolgy, Union Hospital of Tongji Medical University, Wuhan, China, and *Institute of Physiology, München Technical University, Freising-Weihenstephan, Germany

Journal of Huazhong University of Science and Technology 22(1): 69-72.

Summary
Primary open-angle glaucoma (POAG) is a leading cause of blindness, which involves optic neuropathy accompanied  by characteristic visual field defects and is often associated with elevated intraocular pressure due to disturbance of aqueous humor outflow through the trabecular meshwork (TM) (1). The pathophysiology of the TM in POAG has been characterized by an increase in extracellular matrix components and a decrease in the number of TM cells (2). Polypeptide growth factors are critical modulators that control normal cell functions such as proliferation, motility, differentioation, phagocytosis and extracellular matrix synthesis and degradation. Studies of secretion of growth factors which function on TM cells, especially on proliferation and extracellular matrix metabolism, are critical to our understanding of POAG and the development of new antiglaucoma therapy (3). The objective of this study was to use RT-PCR and immunohistochemistry to determine whether IGF-I is expressed by TM cells.

Insulinartige Wachstumsfaktor-1- (IGF-1-)mRNA und IGF-1-Protein 
      Expression in den Zellen des Trabekelwerks im Rinderauge 
  Bovine trabecular meshwork cells express insulin-like growth factor-1 
(IGF-1) mRNA and protein

      Y. Cao (1), M. W. Pfaffl (2), B. Da (1), H. Wei (1)

      (1) Department of Ophthalmology, Union Hospital, Tongji Medical College, 
Huazhong University of Science and Technology, Wuhan, China
      (2) Institut für Physiologie, TU-München, Freising-Weihenstephan

Der Ophthalmologe 2002, 99(7): 555-558

Zusammenfassung

Hintergrund. Gegenstand der Untersuchungen waren die Bestimmungen, ob insulinartige Wachstumsfaktor-1 (IGF-1-)mRNA und das IGF-1-Protein in kultivierten Zellen und in Ex-vivo-Zellen des Trabekelwerks exprimiert werden. 

Methoden. Zum Nachweis der IGF-1-mRNA wurde ein spezifisches, sensitives und zuverlässiges Verfahren angewandt, die reverse Transkription mit folgender Polymerasekettenreaktion (RT-PCR). Das IGF-1-Protein wurde mittels immunhistochemischer Färbung detektiert. 

    Ergebnisse. Die RT-PCR lieferte ein spezifisches 240 bp IGF-1-Produkt, dessen Sequenz absolut homolog der bekannten bovinen Sequenz ist. Das IGF-1-Protein wurde mit einer spezifischen immunhistochemischen Färbung im Zytoplasma der kultivierten Zellen nachgewiesen. 

  Schlussfolgerung. Zusammenfassend ist zu sagen, das die Zellen des Trabekelwerks IGF-1-mRNA sowie das mature IGF-1-Protein exprimieren und in die umliegende Mikroumgebung und in das Augenkammerwasser sekretieren. Das Trabekelwerk wird somit nicht nur durch parakrine, sondern auch durch autokrine Effekte beeinflusst. Ob die Regulation der IGF-1-Produktion zum Krankheitsbild des Weitwinkelglaukoms beiträgt und ob die Förderung der autokrinen IGF-1-Effekte in den Zellen des Trabekelwerks zur Behandlung des grünen Stars beiträgt, ist in weiteren Untersuchungen zu klären. 

    Abstract

    Aim of the study. The purpose of the study was to determine whether cultured bovine trabecular meshwork cells and trabecular tissue ex vivo express insulin-like growth factor-1 (IGF-1) mRNA and protein. 

    Methods. The reverse transcriptase-polymerase chain reaction (RT-PCR) was used for detection of IGF-1 mRNA. To detect the protein on the cells an IGF-1-specific immunohistochemical stain was used on trabecular meshwork cells. 

Results. A single 240 bp RT-PCR product was obtained, the RT-PCR product was verified by sequencing and the derived sequence was homologous to the known bovine sequence. IGF-1 immunostaining was positive in the cytoplasm of trabecular meshwork cells. 

 Conclusions. We conclude that trabecular meshwork cells produce IGF-1 mRNA and contribute to the presence of IGF-1 protein in the trabecular meshwork microenvironment as well as aqueous humor. Trabecular meshwork cells were affected by IGF-1 not only through paracrine but also through autocrine action. Whether regulations in IGF-1 production may contribute to the pathoge-nesis of primary open-angle glaucoma and the possibility of promoting the autocrine action of IGF-1 by trabecular meshwork cells to treat the disease is worth further investigation.

Differences in the somatotropic axis, in blood cortisol, insulin and thyroid hormone concentrations between two pig genotypes
with markedly divergent growth rates and the effects of growth hormone treatment.

Elsaesser, F., Pfaffl, M.W., Meyer, H.H.D., Serpek, B. and Sauerwein, H. (2002)
Animal Science, 74 (3): p423

Summary
The intention of the current study was to gain more insight into the endocrine and molecular control mechanisms of growth in the pig. For this purpose various growth related parameters were determined in 4-month-old barrows of two extreme pig genotyes, the small, obese Göttingen Miniature (GM) and the large and lean German Landrace (DL). Mean growth hormone (GH) concentration, GH pulse frequency and GH pulse amplitude did not differ between breeds. Likewise, plasma IGF-1, thyroxine, tri-iodothyronine (T3) concentrations were similar in both breeds. However the plasma GH response (maximum level and area under curve) to a single i.v. injection of GHRH in DL was higher than in GM (P < 0.05). Furthermore, basal plasma insulin and in particular plasma cortisol concentrations were higher in GM compared with DL pigs (P < 0.05 and P <0.01 respectively). Analysis of cortisol during 4-h frequent blood sampling indicated higher cortisol amplitudes in GM compared with DL (P <  0.01). Specific bGH-binding to hepatic membrane preparations was not different between breeds and IGF-1 m RNA concentrations determined by reverse transcription-polymerase chain reaction in liver, m. semimenbranosus and m. longissimus dorsi were similar in both breeds. I.m. treatment with recombinant
porcine somatotropin (rpST; 70 mg/ kg live weight) over an 8-day period in contemporary barrows increased without any breed difference, plasma IGF-1, T3 and insulin concentrations and hepatic specific bGH-binding, but did not affect thyroxine or cortisol concentrations in plasma. IGF-1 gene expression was also elevated in liver and muscle tissues in rpST-treated animals without obvious breed effects. The observations underline the complexity of the hormonal and molecular control of growth and support the notion that differences in growth potential are the consequence of differences at various levels of the somatotropic axis and apparently relate to differences in other control systems of energy metabolism such as the pituitary adrenal axis or the endocrine pancreas as well.

The mRNA Expression of Insulin Receptor Isoforms (IR-A and IR-B) and IGFR-2 in
the Bovine Corpus Luteum During the Estrous Cycle, Pregnancy, and Induced Luteolysis.

Neuvians TP, Pfaffl MW, Berisha B, Schams D.
Endocrine. 2003 Nov;22(2):93-100.

Institute of Physiology, Technical University Munich, D-85350
Freising-Weihenstephan, Germany.

Isoform A of the human insulin receptor and the IGFreceptor type 2 (IGFR-2) are
both receptors for insulin- like growth factor II (IGF II), which plays a major
role in luteal development and function in bovine species. The objective of this
study was to determine if both insulin receptor isoforms and IGFR-2 were
expressed in the bovine corpus luteum (CL) and if they were regulated during the
estrous cycle, pregnancy, and induced luteolysis. CL were collected at the
slaughterhouse. For induced luteolysis, CL were obtained by transvaginal
ovariectomy at 2, 4, 12, 48, and 64 h after prostaglandin (PG)
F2alpha-injection. Real-time RT-PCR was applied to investigate mRNA expression.
Two alternatively spliced transcripts encoding the insulin receptor were
detected in bovine CL. These two isoforms corresponded to the known isoforms A
(IR-A) and B (IR-B) in humans. IR-A mRNA predominated in bovine CL and was
significantly down-regulated on d 5-7. IR-B mRNA was significantly up-regulated
in the late luteal stage and during early pregnancy. IR-A showed a significant
down-regulation at 48 h after PGF2. IGFR-2 mRNA was significantly up-regulated
in mid and late luteal stages (d 8-18). It is proposed that the differential
mRNA expression of IR-A and IGFR-2, both binding IGF II, may play a role in the
development and function of the bovine corpus luteum.

New sequences of Insulin  receptor subtype mRNA 
at  GENBANK and EMBL

Expression of serotonin receptors:

Quantitative mRNA analysis of bovine 5-HT receptor subtypes in brain, abomasum, and intestine by real-time PCR

Journal of Receptors and Signal Transduction 23(4): 271-287

Serotonin (5-Hydroxytryptamine; 5-HT) is involved in a wide range of physiological functions and pathological states in humans. There exists evidence that serotonergic pathways are also involved in gastrointestinal (GI) motility disorders in ruminants such as displaced abomasum or cecal dilatation/dislocation. This study aimed to develop and validate real-time PCR assays for quantitative mRNA analysis of 5-HT receptor subtypes in bovine tissues. Because the bovine 5-HT receptor nucleotide sequences were completely unknown before, multiple species (human, mouse, and rat) comparisons of nucleotide sequences were done and primers used for bovine cDNA amplification were derived from human or mouse sequences in highly homologous regions. LightCycler real-time PCR assays for the following bovine 5-HT receptor subtypes were developed and validated: 5-HT1a, 5-HT1b, 5-HT1d, 5-HT1e, 5-HT1f, 5-HT2a, 5-HT2b, 5-HT2c, and 5-HT4. Intra- and inter-assay CVs for the 9 established subtypes ranged from 0.49 to 2.54%. As a first physiological application, mRNA expression levels were measured in 3 tissue sample pools of 10 freshly slaughtered, healthy, lactating dairy cows: brain (cortex, thalamus, hypothalamus), abomasum (fundus, corpus, antrum pylori), and intestine pool (ileum, caecum, PLAC, colon). The 5-HT receptor expression was quantified by normalization to the housekeeping gene glyceraldehyde-phosphate-dehydrogenase (GAPDH). The 5-HT receptor expression levels ranged from 0.0001% to 1% 5-HT/GAPDH. There was a high variation of 5-HT receptor subtype mRNA expression within tissue between receptor subtypes and within receptor subtypes between tissues. In conclusion, accurate real-time PCR assays for quantitative analysis of bovine 5-HT receptor subtype gene expression in disease models were developed and validated.

New sequences of 5-HT receptor subtype mRNA 
at  GENBANK and EMBL

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