Fibronectin in the tendon-synovial complex: QuantitationIn vivo andIn vitro by elisa and relative mRNA levels by polymerase chain reaction and northern blot

1994 ◽  
Vol 12 (2) ◽  
pp. 253-261 ◽  
Author(s):  
Brian E. Brigman ◽  
Peiqui Hu ◽  
Hongliang Yin ◽  
Mari Tsuzaki ◽  
W. Thomas Lawrence ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Northern Blot ◽  
Mrna Levels ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Diagnosis of thalassemia using cDNA amplification of circulating erythroid cell mRNA with the polymerase chain reaction [published erratum appears in Blood 1992 Jun 15;79(12):3397]

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2433-2437 ◽  
Author(s):  
SZ Huang ◽  
GP Rodgers ◽  
FY Zeng ◽  
YT Zeng ◽  
AN Schechter
Keyword(s):  
Polymerase Chain Reaction ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Beta Globin ◽  
Chain Reaction ◽  
Globin Mrna ◽  
Gamma Globin ◽  
Polymerase Chain ◽  
Alpha Beta

Abstract We have developed a technique to diagnose the alpha- and beta- thalassemia (thal) syndromes using the polymerase chain reaction to amplify cDNA copies of circulating erythroid cell messenger RNA (mRNA) so as to quantitate the relative amounts of alpha-, beta-, and gamma- globin mRNA contained therein. Quantitation, performed by scintillation counting of 32P-dCTP incorporated into specific globin cDNA bands, showed ratios of alpha/beta-globin mRNA greater than 10-fold and greater than fivefold increased in patients with beta 0- and beta (+)- thal, respectively, as well as a relative increase in gamma-globin mRNA levels. Conversely, patients with alpha-thalassemia showed a decreased ratio of alpha/beta-globin mRNA proportional to the number of alpha- globin genes deleted. This methodology of ascertaining ratios of globin mRNA species provides a new, simplified approach toward the diagnosis of thalassemia syndromes, and may be of value in other studies of globin gene expression at the transcription level.

scholarly journals In vivo and in vitro regulation of erythropoietin mRNA: measurement by competitive polymerase chain reaction

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 617-623 ◽  
Author(s):  
J Fandrey ◽  
HF Bunn
Keyword(s):  
Polymerase Chain Reaction ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Mrna Levels ◽  
Chain Reaction ◽  
Human Hepatoma Cell ◽  
Competitive Polymerase Chain Reaction ◽  
Polymerase Chain

Abstract The regulation of erythropoietin (Epo) production was investigated by competitive polymerase chain reaction, a highly sensitive and accurate means of measuring Epo mRNA levels. Co-amplification of the test sample with added mutant Epo cDNA template corrects for variability in the efficiency of amplification. Epo mRNA levels were determined in tissues of normal rats and in animals with varying degrees of anemia. Reduction of the hematocrit level from 0.40 to 0.15–0.20 resulted in a 300-fold increase in kidney Epo mRNA, which comprised 80% of the total Epo mRNA versus 20% from the liver. In contrast, very low levels detected in lung and spleen were not significantly increased by anemia. The human hepatoma cell line, Hep3B, secretes high levels of Epo in response to hypoxia. This regulation is, to a large extent, transcriptional. When Hep3B cells were incubated in the presence of decreasing O2 tension from 160 to 7 mm Hg, there was a monotonic increase in Epo mRNA to 50 to 100 times the normoxic level. Hyperoxia did not suppress basal expression. When cells were incubated at a PO2 of 7 mm Hg, induction of Epo mRNA was first noted at 30 minutes and was maximal at 5 to 6 hours. After Epo mRNA was boosted by a 4-hour hypoxic incubation, cells were then exposed to normoxia, which shut off further transcription of the Epo gene. The decay of Epo mRNA levels closely followed first order kinetics with a half-life of 2 hours, an effective measurement of message stability.

Cloning and widespread distribution of the rat rod-type cyclic nucleotide-gated cation channel

1997 ◽  
Vol 272 (4) ◽  
pp. C1335-C1344 ◽  
Author(s):  
C. Ding ◽  
E. D. Potter ◽  
W. Qiu ◽  
S. L. Coon ◽  
M. A. Levine ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
In Situ Hybridization ◽  
Pineal Gland ◽  
Northern Blot ◽  
Blot Analysis ◽  
Cyclic Nucleotide ◽  
Northern Blot Analysis ◽  
Chain Reaction ◽  
Polymerase Chain

We used Northern blot analysis, ribonuclease protection assay (RPA), reverse transcriptase-polymerase chain reaction, and in situ hybridization to investigate the hypothesis that the CNG1 isoform of the cyclic nucleotide-gated nonselective cation channel may be widely distributed in tissues of the rat. A cDNA encoding the CNG1 isoform was isolated from rat eye and human retina, and partial sequences were isolated from rat pineal gland and human kidney. Northern blot analysis revealed a 3.1-kilobase (kb) CNG1 transcript in rat eye, pineal gland, pituitary, adrenal gland, and spleen, and a larger transcript of 3.5 kb was found in testis. RPA confirmed the identity of CNG1 mRNA in rat eye, lung, spleen, and brain. Polymerase chain reaction-based detection of the mRNA for CNG1 indicates that the channel is expressed in lower abundance in many other tissues, including thymus, skeletal muscle, heart, and parathyroid gland. The cellular distribution of CNG1 was further studied by in situ hybridization, which demonstrated expression of mRNA in lung, thymus, pineal gland, hippocampus, cerebellum, and cerebral cortex but not in heart or kidney.

scholarly journals Role of type I interferon in the bacillus Calmette-Guérin-induced expression of CXCL10 from human monocytes

2004 ◽  
Vol 13 (5-6) ◽  
pp. 343-348 ◽  
Author(s):  
Patricia Méndez Samperio ◽  
Artemisa Trejo ◽  
Elena Miranda
Keyword(s):  
Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Method ◽  
Type I ◽  
Mrna Levels ◽  
Human Monocytes ◽  
Bacillus Calmette Guerin ◽  
Chain Reaction ◽  
Reaction Method ◽  
Polymerase Chain

BACKGROUND: The proinflammatory chemokine CXCL10, in addition to its chemotactic properties, is also involved in the stimulation of natural killer and T-cell migration inMycobacterium tuberculosisinfection. In this study, our experiments were designed to determine the role of interferon (IFN)-αβ in the production of CXCL10 by human monocytes infected withMycobacterium bovisbacillus Calmette-Guérin (BCG).Methods: The concentrations of CXCL10 in culture supernatants of monocytes infected withM. bovisBCG were determined by enzyme-linked immunosorbent assay. CXCL10 mRNA levels were determined by the reverse transcription-polymerase chain reaction method.Results: We have shown the induction of CXCL10 following infection withM. bovisBCG in a dose-dependent and time-dependent manner. Importantly, the secretion of CXCL10 in response toM. boviswas increased by IFN-α. These results were further confirmed by the fact that the addition of an anti-IFN-αβ neutralizing antibody completely reversed the stimulatory effect, whereas an isotype-matched control antibody had no significant effect on CXCL10 secretion. It is important to note that no significant effect of type I IFN on CXCL8 production inM. bovis-infected monocytes was observed. This was consistent with the finding by the reverse transcription-polymerase chain reaction method that treatment with anti-IFN-α/β antibodies potentially inhibited CXCL10 mRNA levels, whereas no significant effect was observed on CXCL8 mRNA. Moreover, in THP-1 monocytes and THP-1 macrophages, the addition of exogenous IFN-α stimulated CXCL10 secretion.Conclusions: Collectively, these results indicate that the type I IFN may play an important role to modulate the expression of CXCL10 inM. bovisBCG infection. Studies onM. bovis-induced chemokine secretion could provide important insight into the regulation of the immune response against tuberculosis.

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