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PubMed2000

Poly(A) cDNA-specific (PACS) RT-PCR: a quantitative method for the measurement of any poly(A)-containing mRNA not affected by contaminating genomic DNA.

We present a simple and efficient RT-PCR method for the detection and quantitation of any poly(A)-containing mRNA that is not affected by contaminating genomic DNA and does not rely on exhaustive DNase digestion protocols. The technique described here requires the use of an antisense primer designed to contain 6-8 bp cDNA-specific sequence and an additional 17 Ts located on the 5' end to take advantage of the poly(A) tail. A second cDNA-specific sense primer can be used that does not need to be separated by intronic DNA sequence.

Animals↗
PubMed1998

Utility of major leukocyte subpopulations for monitoring secondary cytomegalovirus infections in renal-allograft recipients by PCR.

The feasibility of the major peripheral blood leukocyte (PBL) subsets for use in qualitative and quantitative PCR to monitor secondary cytomegalovirus (CMV) infection and ganciclovir therapy was assessed with 188 blood samples derived from 40 CMV immunoglobulin G-positive renal-allograft recipients. In pp65 antigen-positive patients all leukocyte fractions, but only 79.5% of plasma preparations, were PCR positive. In pp65 antigen-negative samples from patients after antiviral treatment only 7.3% of polymorphonuclear cell (PMNL) samples, but 81.8% of peripheral blood mononuclear cells (PBMC), and 10.9% of plasma samples remained PCR positive. Similarly, in patients with latent infections only 5.0% of PMNL, but 51.7% of PBMC preparations, and 8.0% of plasma samples were PCR positive. Regarding patients with active CMV infection, CMV DNA copy numbers in PMNL correlated significantly with pp65 antigen-positive cell counts before and after onset of ganciclovir therapy. Significant differences in CMV DNA copy numbers in PMNL and plasma were observed (i) between patients with symptomatic infection and those with asymptomatic infection and (ii) between patients with active infection and those with latent infection. In contrast, PBMC harbored equally low CMV DNA levels both in patients with active infection and those with latent infections, and no decline of CMV DNA load in PBMC was observed during antiviral treatment. We conclude that detection of CMV DNA in PMNL, not in PBMC, is associated with active infections and is more sensitive than detection of CMV DNA in plasma. Negative PCR results for PMNL after antiviral therapy indicate recovery, and fewer unwanted positive results occur compared to PBMC and plasma. Therefore, purified PMNL should be preferred for analysis by qualitative CMV PCR to avoid unwanted positive results. The CMV DNA load in PBMC compared with that in PMNL is negligible during active infection, so mixed PBL are sufficient for use in quantitative PCR.

Cytomegalovirus↗
PubMed2001

Quantitative PCR-ELAHA for the determination of retroviral vector transduction efficiency.

Current methods to detect transduction efficiency during the routine use of integrating retroviral vectors in gene therapy applications may require the use of radioactivity and usually rely upon subjective determination of the results. We have developed two competitive quantitative assays that use an enzyme-linked, amplicon hybridization assay (ELAHA) to detect the products of PCR-amplified regions of transgene from cells transduced with Moloney murine leukemia virus vectors. The quantitative assays (PCR-ELAHA) proved to be simple, rapid, and sensitive, avoiding the need for Southern hybridization, complex histochemical stains, or often subjective and time-consuming tissue culture and immunofluorescence assays. The PCR-ELAHA systems can rapidly detect proviral DNA from any retroviral vector carrying the common selective and marker genes neomycin phosphotransferase and green fluorescent protein, and the methods described are equally applicable to other sequences of interest, providing a cheaper alternative to the evolving real-time PCR methods. The results revealed the number of copies of retrovector provirus present per stably transduced cell using vectors containing either one or both qPCR targets.

Animals↗
PubMed2005

Comparison of the SYBR Green and the hybridization probe format for real-time PCR detection of HHV-6.

A comparative study was conducted of a novel real-time quantitative PCR test (LightCycler System) with FastStart DNA Master(PLUS) SYBR Green I dye to detect DNA of human herpes virus 6 (HHV-6). Results were compared with those of a real-time quantitative PCR with hybridization probe (HP) formats using the fluorescence resonance energy transfer method, and with those of a single qualitative PCR test. The detection limit of the test with SYBR Green I dye was 20 copies of the virus, similar to that of the other two tests. The reproducibility was satisfactory. The new test has the same advantages as real-time PCR with HP formats and offers a greater versatility at lower cost.

Benzothiazoles↗
PubMed2003

CMV infection of the renal allograft is much more common than the pathology indicates: a retrospective analysis of qualitative and quantitative buffy coat CMV-PCR, renal biopsy pathology and tissue CMV-PCR.

BACKGROUND: Quantitative blood polymerase chain reaction (PCR) for cytomegalovirus (CMV) is used to direct therapy in kidney transplant patients, but cytomegalic inclusions are rarely found in allograft renal biopsies even with an elevated serum creatinine and apparent CMV disease. The relationship between quantitative blood CMV and renal allograft pathology is unknown. METHODS: Thirteen biopsy samples were available for analysis from patients suspected of CMV disease, who had a buffy coat CMV-PCR drawn within 2-5 days of a renal allograft biopsy for an elevated creatinine. All were evaluated for CMV pathologically, by light microscopy, immunohistochemistry, in situ hybridization and tissue PCR. RESULTS: Qualitative and quantitative buffy coat CMV-PCR were positive in 10/13 (77%) patients. Tissue CMV-PCR was positive in five (50%) biopsies, including two with CMV inclusions and three with no inclusions. Quantitative buffy coat CMV-PCR levels did not correlate with detection of CMV inclusions in renal tissue. Paradoxically, quantitative buffy coat CMV-PCR was low (239 and 538 copies/microg of DNA) when CMV inclusions were detected. All five biopsies with acute rejection were associated with CMV viraemia and two of the five with allograft CMV inclusions. A quantitative buffy coat CMV-PCR of <100 copies/microg of DNA ruled out disease with CMV inclusions. CONCLUSIONS: CMV nephropathy is much more common than previously reported when sensitive techniques are used for detection in tissue. Acute rejection and CMV viraemia occur commonly together in patients at risk for CMV. Quantitative buffy coat CMV-PCR does not correlate with the presence of CMV inclusions. These findings have implications for management of patients who have elevated serum creatinine and are at risk for CMV disease.

Biopsy↗
PubMed2006

Design, development and evaluation of a competitive RT-PCR for quantitation of GBV-C RNA.

Although GB virus C (GBV-C) hepatocyte pathogenicity is still controversial, it appears that at least some strains of this virus are lymphotropic. During the past few years, several reports have documented an apparently beneficial role played by GBV-C in the course of HIV-1 infection. At present, a commercial kit for GBV-C RNA quantitation is not available. In this study, a competitive RT-PCR method for GBV-C in serum samples is described. The sensitivity of the assay proved to be 10(4) and 10(3) genomic equivalents for positive and negative sense RNAs, respectively. This method will discriminate specifically between positive and negative strand RNAs with a discrimination index of at least five log10. Out of 60 samples from different hematological disorders (n = 49), HIV-1 positive patients (n = 7), and blood donors (n = 4), 10 proved to be GBV-C RNA positive. Viral load ranged from 1.1 x 10(7) to 2.34 x 10(8) genomic equivalents/ml. Such values correlated linearly (r = 0.986) with those obtained by a 10-fold serial dilution method. In studies exploring the GBV-C pathogenicity, the measurement of viral load may contribute to understand the possible mechanisms involved.

GB virus C↗
PubMed2001

Recovery efficiences on nucleic acid extraction kits as measured by quantitative LightCycler PCR.

AIMS: To compare the efficiency of five nucleic acid extraction kits for processing clinical material for the diagnosis of infection. METHODS: Five nucleic acid extraction kits for processing clinical material for the diagnosis of infection were compared for their relative efficiencies in purifying and recovering either viral DNA or RNA from serum samples. Quantitative polymerase chain reaction (PCR) assays for hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA were performed on the Light-Cycler instrument to determine the relative concentrations of the viral nucleic acids recovered by the various protocols. RESULTS: Large differences between kits in recovery efficiencies were observed for HBV DNA, with those protocols using enzyme digestion in addition to chaotropic solutions performing better than those using chemical disruption alone. CONCLUSIONS: There were large differences between the kits and it appeared that those extraction kits containing a proteolytic enzyme and carrier nucleic acid (and that are supplied RNase free) have the widest potential application in the routine microbiology laboratory.

DNA, Viral↗
PubMed2005

N- and C-terminal isoforms of Arg quantified by real-time PCR are specifically expressed in human normal and neoplastic cells, in neoplastic cell lines, and in HL-60 cell differentiation.

The human ABL2 (or ARG) gene codes for a nonreceptor tyrosine kinase is involved in translocation with the ETV6 gene in human leukemia and has an altered expression in several human carcinomas. Two isoforms of Arg with different N-termini (1A and 1B) have been described. The C-terminal domain of Arg contains two F-actin-binding sequences that perform a number of actions related to cell morphology and motility by interacting with actin filaments. We have identified different-sized specific cDNAs in hematopoietic, epithelial, nervous, and fibroblastic cells by means of the reverse transcription (RT)-polymerase chain reaction (PCR) analysis of human Arg mRNA. Some of these cDNAs showed an adjunctive alternative splice event involving the 63 bp sequence of exon II, thus leading to four cDNA types with different N-termini: 1A long and short, and 1B long and short. Other cDNAs lacked a 309 bp sequence in the last exon involving one of the C-terminal F-actin binding domains, thus giving rise to two cDNA types: C-termini long and short. Quantified by real-time PCR-quantitative RT-PCR-these Arg transcript isoforms have specific expression patterns not only in different normal and tumor cell types, but also during cell differentiation and growth arrest. These isoforms maintained the open reading frames, and eight putative proteins were predicted. The different C-termini isoforms seem to retain the same quantitative reciprocal ratio of their respective transcripts. The Arg protein isoforms with different C-terminal actin-binding domains and different N-termini might have specific cellular localizations/concentrations, and differently regulated catalytic activity with different implications in normal and neoplastic cells.

Amino Acid Sequence↗
PubMed2000

Real-time quantitation of bcr-abl transcripts in haematological malignancies.

We have applied an automated real-time quantitative PCR assay using a double-labeled fluorogenic probe to detect t(9;22)-positive cells in haematological malignancies. The results are expressed as the ratio of chimeric bcr-abl transcripts on abl transcripts. Highly reproducible results were obtained for t(9;22)-positive K562 RNA. Ten copies of bcr-abl DNA from a recombinant KW-3 plasmid and one positive cell in 10(4) can be detected. Thirty-two patients with chronic myeloid leukaemia (CML), 25 with acute leukaemia, 12 with myelodysplastic syndromes and 7 with other myeloproliferative syndromes were tested. Follow-up data were obtained in bcr-abl positive cases. Results were compared with those of conventional nested RT-PCR and cytogenetics. Real-time quantitative RT-PCR values correlated well with both these methods. However, in some cases the only means of detecting early relapse or blastic transformation was to examine the kinetics of real-time quantitative RT-PCR. Thus, real-time quantitative RT-PCR appears suitable for the diagnosis and follow-up of patients with the t(9;22) translocation.

Acute Disease↗
PubMed2001

Nested quantitative real time PCR for detection of occult tumor cells.

Quantification of tumor mRNA markers expressed by occult circulating tumor cells may be of prognostic value in a variety of neoplasms and disease stages. We therefore developed a novel real time nested polymerase chain reaction (PCR) assay to quantify rare transcripts using the light cycler system. Tyrosinase mRNA expressed by melanocytes and melanoma cells was used as a model. Ten-milliliter samples of ethylenediaminetetra-acetate (EDTA) blood from healthy volunteers were spiked with 10-10(4) RVH melanoma cells. Following RNA extraction and cDNA synthesis, a nested PCR with specific primers was performed. Resonance energy transfer between fluorescently labeled hybridization probes specific for the amplified portion of the tyrosinase transcript allowed continuous monitoring of the second round of PCR. The number of preamplification cycles in the first round was varied between 20 and 35. Our results show that nested PCR revealed quantitative data, regardless of the number of cycles used in the first round. The range of real time data can span four logs of target mRNA concentrations (1-10(3) tumor cells/ml of blood) and the x-intercepts of the log phases were always consistent with the amount of transcripts in the sample. These experiments indicate that nested PCR, which is much more sensitive than single-round PCR, is a useful tool for the quantification of rare transcripts--a result with important implications for the study of minimal residual disease. The assay is currently being adapted for other tumor types including leukemias as well as other solid tumors.

DNA Primers↗
PubMed2002

Real-time PCR for quantification of human herpesvirus 6 DNA from lymph nodes and saliva.

A real-time quantitative PCR assay has been developed to measure human herpesvirus 6 (HHV-6) DNA in biological specimens. The assay sensitivity was 10 copies of DNA per well, with a linear dynamic range of 10 to 10(7) copies of HHV-6 DNA. Intra- and interassay variations were, respectively, 0.88 and 0.8% for samples containing 10(2) DNA copies, 0.99 and 0.96% for samples containing 10(4) copies, and 0.76 and 0.9% for samples containing 10(6) copies. Among 34 saliva samples from healthy subjects, 26 were found to contain HHV-6 DNA (76.5%; median, 23,870 copies/ml), and following a single freeze-thaw cycle, 25 of the same samples were found to be positive for HHV-6 DNA, although at a statistically significantly lower concentration (median, 3,497 copies/ml). The assay enabled detection of HHV-6 DNA in lymph node biopsies from patients with Hodgkin's disease (HD) (13 of 37 patients [35.1%]), B-cell neoplasms (8 of 36 patients [22.2%]), and T- or NK-cell neoplasms (3 of 13 patients [23.1%]), with concentrations ranging from 100 to 864,640 HHV-6 copies per microg of DNA (HHV-6B being found in every case except two). All HD patients infected with HHV-6 presented clinically with the nodular sclerosis subtype of HD. The real-time quantitative PCR assay developed here was simple to perform and was sensitive over a wide range of HHV-6 concentrations. It therefore appears to be of potential value in clinical investigation or diagnosis of HHV-6 infection.

Adult↗
PubMed2006

Application of real-time PCR for quantitative detection of Vibrio parahaemolyticus from seafood in eastern China.

Vibrio parahaemolyticus is recognized as a leading human food-borne pathogen. A TaqMan PCR assay based on the gyrase B gene (gyrB) sequence of V. parahaemolyticus was developed for quantitative detection of V. parahaemolyticus in seafood. The study involving 27 V. parahaemolyticus and 10 strains of other species indicated that the real-time PCR test was highly specific. The sensitivity of the assay was approximately a single CFU per PCR in pure culture and six to eight CFU per PCR in spiked raw oyster, respectively. Real-time PCR values of artificially inoculated oyster homogenates correlated well with plate counts determined using culture methods. A total of 300 seafood samples were analyzed and 78 (26%) of these samples were positive for V. parahaemolyticus using a conventional culture method and 97 (32.3%) using the real-time PCR assay. All culture-positive samples were PCR-positive. However, 19 samples positive by PCR were culture-negative. The results show that retail seafood is commonly contaminated with V. parahaemolyticus in harvest season in eastern China. These data also indicate that real-time PCR can provide sensitive species-specific detection and quantification of V. parahaemolyticus in seafood without prior isolation and characterization of the bacteria by traditional microbiological methods.

Animals↗
PubMed1998

Measurement of tryptophan hydroxylase mRNA levels by competitive RT-PCR.

Tryptophan hydroxylase (TPH) is the rate limiting enzyme in serotonin biosynthesis [D.G. Grahame-Smith, Tryptophan hydroxylation in brain, Biochem. Biophys. Res. Commun. 16 (1964) 586-592 [19]]. As such, the TPH gene is a likely target for modulation of serotonergic function, which has been associated with several psychiatric disorders [E.C. Azmitia, P.M. Whitaker-Azmitia, Awakening the sleeping giant: anatomy and plasticity of the brain serotonergic system, J. Clin. Psychiatry 52 (12, Suppl.) (1991) 4-16 [1]; R.P. Hart, R. Yang, L.A. Riley., T.L. Green, Post-transcriptional control of tryptophan hydroxylase gene expression in rat brain stem and pineal gland, Mol. Cell. Neurosci. 2 (1991) 71-77 [20]; M.J. Owens, C.B. Numeroff, Role of serotonin in the pathophysiology of depression: focus on the serotonin transporter, Clin. Chem. 40 (1994) 288-295 [24]]. Unfortunately, it has been technically difficult to measure TPH mRNA levels in central serotonergic neurons due to its low levels. For example, detection with ribonuclease protection assays requires pooling of 5-10 dissected brainstems [M.C. Darmon, B. Guibert, V. Leviel, M. Ehret, M. Maitre, J. Mallet, Sequence of two mRNAs encoding active rat tryptophan hydroxylase, J. Neurochem. 51 (1988) 312-316 [15]; B.L. Jacobs, E.C. Azmitia, Structure and function of the brain serotonin system, Physiol. Rev. 72 (1992) 165-229 [21]]. This protocol describes the use of competitive RT-PCR to measure TPH mRNA levels from rat brain. First described in 1988, competitive RT-PCR has become an accepted method of measuring RNA abundance [M. Clementi, S. Menzo, P. Bagnarelli, A. Manzin, A. Valenza, P.E. Varaldo, Quantitative PCR and RT-PCR in virology, PCR Methods Appl. 2 (1994) 191-196 [12]; N.C.P. Cross, Quantitative PCR techniques and applications, Br. J. Haematol. 89 (1995) 693-697 [14]; K.P. Foley, M.W. Leonard, J.D. Engel, Quantitation of RNA using the polymerase chain reaction, Trends Genet. 9 (1993) 380-385 [17]; P.D. Siebert, J.W. Larrick, Competitive PCR, Nature 359 (1992) 558 [27]]. Competitive RT-PCR uses co-amplification with a known quantity of an in vitro transcribed RNA which amplifies using the same primers and thus competes for reactants with the product of interest. As the two products amplify with the same efficiency, the relative abundance of the two amplification products remains constant, and thus can be used to determine initial tissue TPH mRNA levels [G. Gilliland, S. Perrin, K. Blanchard, H.F. Bunn, Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction, Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 2725-2729 [18]; A.M. Wang, M. V. Doyle, D.F. Mark, Quantitation of mRNA by the polymerase chain reaction, Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 9717-9721 [31]]. We first demonstrate equivalent results between RNA slot blots and competitive RT-PCR using the CA77 thyroid C cell line [M.S. Clark, A. F. Russo, Tissue-specific glucocorticoid regulation of tryptophan hydroxylase mRNA levels, Mol. Brain Res. 48 (1997) 346-354 [9]]. We then describe the use of competitive RT-PCR to measure TPH mRNA levels in RNA isolated from rat brain poly-A+ RNA.

Animals↗
PubMed1992

Rapid quantitation by PCR of endomycorrhizal fungi colonizing roots.

The VANS1/NS21 primer pair is useful for specifically amplifying a 550-bp ribosomal (r) DNA fragment from arbuscular endomycorrhizal fungi, directly from colonized root extracts. A procedure to quantitate these obligatory biotrophs rapidly, based on competitive PCR, was developed by constructing a suitable internal standard to be used with these primers. A 130-bp deletion in the Glomus mossae VANS1/NS21 amplified rDNA fragment was produced by amplifying separately external portions of that fragment, followed by ligation and amplification using the original external primers. When this deleted fragment was added to G. mossae rDNA, amplification using VANS1/NS21 primers yielded the two expected products of 430 bp and 550 bp, respectively, resolved by agarose electrophoresis. This fragment was cloned into the pCL1920 plasmid, a low-copy-number vector (five copies per cell), and mixed with the roots to be analyzed. This provides for a rapid quantitative assay because both steps--extraction of DNA from colonized roots and PCR amplification--are taken into account by the same internal standard. Using this procedure, a sample of colonized leek roots (Allium porum x Glomus vesiculiferum) was shown to contain 5 x 10(4) copies of arbuscular endomycorrhizal fungi rDNA genes per milligram of fresh weight.

Allium↗
PubMed2002

Identification, detection, and enumeration of human bifidobacterium species by PCR targeting the transaldolase gene.

Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations.

Base Sequence↗
PubMed2002

Kinetic characterisation of primer mismatches in allele-specific PCR: a quantitative assessment.

A novel method of estimating the kinetic parameters of Taq DNA polymerase during rapid cycle PCR is presented. A model was constructed using a simplified sigmoid function to represent substrate accumulation during PCR in combination with the general equation describing high substrate inhibition for Michaelis-Menten enzymes. The PCR progress curve was viewed as a series of independent reactions where initial rates were accurately measured for each cycle. Kinetic parameters were obtained for allele-specific PCR (AS-PCR) amplification to examine the effect of mismatches on amplification. A high degree of correlation was obtained providing evidence of substrate inhibition as a major cause of the plateau phase that occurs in the later cycles of PCR.

Alleles↗
PubMed2004

Refined quantitative fluorescent PCR of Y-chromosome DNA sequences mosaics in Turner's syndrome patients--alternative to real-time PCR.

BACKGROUND: Real-time polymerase chain reactions (PCRs) are the most frequently used techniques for gonosomal mosaics quantification. The primary aim of this work is to assess and optimize the refined technique of quantitative fluorescent polymerase chain reaction (RQF PCR) in the quantification of Y-chromosome sequences in gonosomal mosaics. The method was applied to the analysis of Y-chromosome sequences (amelogenin gene, AMELX/Y-loci) in peripheral lymphocytes and gonadal tissues in Y-positive Turner's syndrome (TS) patients. METHODS: RQF PCR was used for molecular quantification, and fluorescent in situ hybridization (FISH) technique was used for comparison. RESULTS: Based on a formulated calibration curve, DNA mosaics from six Y-positive patients and gonads from one patient were deducted. For calculation of rare mosaics, it is possible to take advantage of a new empirical formula. FISH results were comparable to RQF PCR. CONCLUSION: The sensitivity of RQF PCR brings significant progress in the analysis of gonosomal aberrations. RQF PCR also finds applications in prenatal diagnostics of maternal contaminations of amniotic fluid and foetal DNA in maternal blood and analysis of chimerism in patients after bone marrow transplantation. The method is very convenient for determining the number of testis-specific protein, Y-linked (TSPY) gene repetitions.

Calibration↗
PubMed2007

Insulin expression in the brain and pituitary cells of tilapia (Oreochromis niloticus).

While the presence of immunoreactive insulin in the central nervous system of many vertebrate species is well known, the origin of brain insulin is still debated. In this study, we applied RT-PCR, quantitative RT-PCR (qRT-PCR), and Northern hybridization to examine expression of the insulin gene in different tissues of an adult teleost fish, the Nile Tilapia (Oreochromis niloticus). We found that the insulin gene is transcribed at a high level in Brockmann bodies (pancreatic islet organs) and at a low level in the brain and pituitary gland. In the brain, insulin transcripts were detected in all areas by qRT-PCR and in situ hybridization. The highest level of insulin mRNA was found in the hypothalamus. The level of insulin transcription in the pituitary gland was 6-fold higher than that in the brain and 4.6-fold higher than that in the hypothalamus. Furthermore, insulin mRNA and immunoreactive insulin-like protein was detected in the pituitary gland using in situ hybridization, immunohistochemistry, and Western blot analysis. Our results indicate that in adult tilapia insulin expression is not restricted to the endocrine pancreatic cells, but also occurs in endocrine cells of the pituitary gland and in the neuronal cells of the brain, suggesting that the brain/pituitary gland might represent extrapancreatic origin of insulin production.

Animals↗