RT-PCR & real-time RT-PCR applications in Molecular Endocrinology
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,
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.
objective of the study was to demonstrate the mRNA expression of
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
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
The mRNA expression of ERalpha and ERbeta in granulosa cells (GC)
RNA) increased continuously during follicle growth but without any
change. The expression of mRNA for PR in follicles (lower fg/microg
increased continuously to maximum level in preovulatory follicles with a
change only in TI. The highest mRNA expression for ERalpha
RNA) was detected in corpus luteum (CL) during the early luteal
following by a significant decrease of expression during the mid, late,
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.
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
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.
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)
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
Pfaffl MW, Hocquette JF, Gerrard DE, Picard B, Geay Y, Sauerwein H. (
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.
objective of this study was to compare
androgen receptor (AR) expression in three
different muscles of bulls and
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
Quantification of androgen receptor mRNA in tissues by competitive co-amplification of a template in RT-PCR
Sauerwein H, Pfaffl MW, Meyer HH. (1996)
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
product yield. To overcome these tube-to-tube differences
efficiency, an internal control AR cRNA was reverse transcribed along with the
target mRNA using the same primers. This standard was
Expression of estrogen and androgen receptor in the bovine gastrointestinal tract
Pfaffl M.; Hagen-Mann K.; Malucelli A. & Meyer H.H.D. (1995)
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
stage as demonstrated earlier for the ER protein, indicating that there might be
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
presence of ER and androgen receptor (AR)
Uterine Androgen Receptor mRNA Expression in Metestrous and
Anestrous Bitches being healthy or suffering from pyometra
Brandstetter, M.W. Pfaffl, H.H.D. Meyer, E. Möstl, J. Handler
& K. Arbeiter (1998)
determination of environmental androgenic/antiandrogenic compounds
A. Hartel, A.
Didier, M.W. Pfaffl, Heinrich H.D. Meyer
receptor subtypes in the mammary gland of dairy cows
T. Inderwies, M. W.
Pfaffl, H. H. D. Meyer, J. W. Blum &
R. M. Bruckmaier
sequences of alpha-adrenergic receptor subtype mRNA
their relation to adrenergic receptor mRNA expression
Inderwies, M. W. Pfaffl & R. M.
Stimulation of - and -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 - and -adrenergic receptor stimulation were evaluated after application of an - and -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 - and -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 1-, 2-, and 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 2-adrenergic receptor binding (maximal binding capacity, Bmax) and positively correlated with 2-adrenergic receptor expression at the mRNA level (crossing point (CP) of the real-time PCR). During -adrenergic receptor stimulation, there was a positive correlation between milkability and 2-adrenergic receptor mRNA expression, whereas during -adrenergic receptor stimulation no correlations were detected. Dose–response relationships were existing during -adrenergic receptor stimulations, but not during -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 -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)
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
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
insulin was highest (P < 0.05) in GrPo
immediately after birth and was primarily seen in the
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
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
at different intestinal sites and in intestine and liver
changed during the perinatal period.
Sauter SN, Ontsouka E, Roffler B, Zbinden Y, Philipona C, Pfaffl MW, Breier BH, Blum JW, Hammon HM (2003)
and Physiology, University of Berne, Institute of
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.
I P Georgiev, T M Georgieva, M Pfaffl, H M Hammon and J W Blum (2003)
Journal of Endocrinology, 176: 121-132
development and validation of an internally standardised competitive RT-PCR
Pfaffl M, Meyer
HH, Sauerwein H. (1998)
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
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)
in bovine trabecular Meshwork tissue and cells
Yang Cao, Houren
Wei, Da Banghong, Michael W. Pfaffl & Li Zhongyu (2002)
Huazhong University of Science and Technology 22(1): 69-72.
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,
2002, 99(7): 555-558
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.
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.
in the somatotropic axis, in blood cortisol, insulin and
thyroid hormone concentrations between two pig
Pfaffl, M.W., Meyer, H.H.D., Serpek, B. and Sauerwein, H. (2002)
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
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.
sequences of 5-HT receptor subtype mRNA