Demonstration of neurotensin messenger RNA in PC12 pheochromocytoma cells using synthetic oligonucleotides for hybridization and polymerase chain reaction

Y. Tsutsumi, Philip Stork, A. S. Tischler, H. J. Wolfe

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

The aim of this study was to evaluate the applicability of synthetic oligonucleotides to the study of the expression and regulation of neuropeptide messenger RNA in neuroendocrine cells. As a model system, neurotensin gene expression was studied in the rat pheochromocytoma cell line, PC12, which demonstrates cumulative increases in neurotensin production in response to combinations of nerve growth factor, dexamethasone, forskolin and lithium. DNA oligonucleotide probes complementary to the rat neurotensin messenger RNA sequence were synthesized and labeled with [32P] by T4 polynucleotide kinase and terminal deoxynucleotidyl transferase. In Northern blot analysis, two mRNA bands of 10 kb and 15 kb were visible in total RNA preparations of the stimulated PC12 cells but not in the unstimulated cell extract. In situ hybridization histochemistry showed a strong signal in about 30% of the stimulated PC12 cells. The tailed probe gave a stronger signal than the 'kinased' probe. The tailed oligonucleotide probe with a sense sequence did not show any hybridization. The unstimulated PC12 cells were unlabeled by either probe. The polymerase chain reaction (PCR) using the same oligonucleotides (with sense and antisense directions) as primer pairs specifically amplified the neurotensin sequence in the stimulated PC12 cell RNA after it was reverse-transcribed into complementary DNA: a low level of neurotensin gene message was also detected in the unstimulated PC12 cells after 30 cycles of the PCR. Our findings suggest that oligonucleotides play a role in both quantitative and morphological studies of neuroendocrine expression at the cellular level.

Original languageEnglish (US)
Pages (from-to)1-9
Number of pages9
JournalBiomedical Research
Volume11
Issue number1
StatePublished - 1990
Externally publishedYes

Fingerprint

Neurotensin
Polymerase chain reaction
PC12 Cells
Pheochromocytoma
Oligonucleotides
Demonstrations
Polymerase Chain Reaction
Messenger RNA
Oligonucleotide Probes
Rats
Polynucleotide 5'-Hydroxyl-Kinase
RNA
DNA Nucleotidylexotransferase
Nerve Growth Factor
Colforsin
Neuroendocrine Cells
Neuropeptides
Lithium
Gene expression
Dexamethasone

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Demonstration of neurotensin messenger RNA in PC12 pheochromocytoma cells using synthetic oligonucleotides for hybridization and polymerase chain reaction. / Tsutsumi, Y.; Stork, Philip; Tischler, A. S.; Wolfe, H. J.

In: Biomedical Research, Vol. 11, No. 1, 1990, p. 1-9.

Research output: Contribution to journalArticle

@article{fb6fe13b2d354406a8285aaa9efa4e8a,
title = "Demonstration of neurotensin messenger RNA in PC12 pheochromocytoma cells using synthetic oligonucleotides for hybridization and polymerase chain reaction",
abstract = "The aim of this study was to evaluate the applicability of synthetic oligonucleotides to the study of the expression and regulation of neuropeptide messenger RNA in neuroendocrine cells. As a model system, neurotensin gene expression was studied in the rat pheochromocytoma cell line, PC12, which demonstrates cumulative increases in neurotensin production in response to combinations of nerve growth factor, dexamethasone, forskolin and lithium. DNA oligonucleotide probes complementary to the rat neurotensin messenger RNA sequence were synthesized and labeled with [32P] by T4 polynucleotide kinase and terminal deoxynucleotidyl transferase. In Northern blot analysis, two mRNA bands of 10 kb and 15 kb were visible in total RNA preparations of the stimulated PC12 cells but not in the unstimulated cell extract. In situ hybridization histochemistry showed a strong signal in about 30{\%} of the stimulated PC12 cells. The tailed probe gave a stronger signal than the 'kinased' probe. The tailed oligonucleotide probe with a sense sequence did not show any hybridization. The unstimulated PC12 cells were unlabeled by either probe. The polymerase chain reaction (PCR) using the same oligonucleotides (with sense and antisense directions) as primer pairs specifically amplified the neurotensin sequence in the stimulated PC12 cell RNA after it was reverse-transcribed into complementary DNA: a low level of neurotensin gene message was also detected in the unstimulated PC12 cells after 30 cycles of the PCR. Our findings suggest that oligonucleotides play a role in both quantitative and morphological studies of neuroendocrine expression at the cellular level.",
author = "Y. Tsutsumi and Philip Stork and Tischler, {A. S.} and Wolfe, {H. J.}",
year = "1990",
language = "English (US)",
volume = "11",
pages = "1--9",
journal = "Biomedical Research",
issn = "0388-6107",
publisher = "Biomedical Research Foundation",
number = "1",

}

TY - JOUR

T1 - Demonstration of neurotensin messenger RNA in PC12 pheochromocytoma cells using synthetic oligonucleotides for hybridization and polymerase chain reaction

AU - Tsutsumi, Y.

AU - Stork, Philip

AU - Tischler, A. S.

AU - Wolfe, H. J.

PY - 1990

Y1 - 1990

N2 - The aim of this study was to evaluate the applicability of synthetic oligonucleotides to the study of the expression and regulation of neuropeptide messenger RNA in neuroendocrine cells. As a model system, neurotensin gene expression was studied in the rat pheochromocytoma cell line, PC12, which demonstrates cumulative increases in neurotensin production in response to combinations of nerve growth factor, dexamethasone, forskolin and lithium. DNA oligonucleotide probes complementary to the rat neurotensin messenger RNA sequence were synthesized and labeled with [32P] by T4 polynucleotide kinase and terminal deoxynucleotidyl transferase. In Northern blot analysis, two mRNA bands of 10 kb and 15 kb were visible in total RNA preparations of the stimulated PC12 cells but not in the unstimulated cell extract. In situ hybridization histochemistry showed a strong signal in about 30% of the stimulated PC12 cells. The tailed probe gave a stronger signal than the 'kinased' probe. The tailed oligonucleotide probe with a sense sequence did not show any hybridization. The unstimulated PC12 cells were unlabeled by either probe. The polymerase chain reaction (PCR) using the same oligonucleotides (with sense and antisense directions) as primer pairs specifically amplified the neurotensin sequence in the stimulated PC12 cell RNA after it was reverse-transcribed into complementary DNA: a low level of neurotensin gene message was also detected in the unstimulated PC12 cells after 30 cycles of the PCR. Our findings suggest that oligonucleotides play a role in both quantitative and morphological studies of neuroendocrine expression at the cellular level.

AB - The aim of this study was to evaluate the applicability of synthetic oligonucleotides to the study of the expression and regulation of neuropeptide messenger RNA in neuroendocrine cells. As a model system, neurotensin gene expression was studied in the rat pheochromocytoma cell line, PC12, which demonstrates cumulative increases in neurotensin production in response to combinations of nerve growth factor, dexamethasone, forskolin and lithium. DNA oligonucleotide probes complementary to the rat neurotensin messenger RNA sequence were synthesized and labeled with [32P] by T4 polynucleotide kinase and terminal deoxynucleotidyl transferase. In Northern blot analysis, two mRNA bands of 10 kb and 15 kb were visible in total RNA preparations of the stimulated PC12 cells but not in the unstimulated cell extract. In situ hybridization histochemistry showed a strong signal in about 30% of the stimulated PC12 cells. The tailed probe gave a stronger signal than the 'kinased' probe. The tailed oligonucleotide probe with a sense sequence did not show any hybridization. The unstimulated PC12 cells were unlabeled by either probe. The polymerase chain reaction (PCR) using the same oligonucleotides (with sense and antisense directions) as primer pairs specifically amplified the neurotensin sequence in the stimulated PC12 cell RNA after it was reverse-transcribed into complementary DNA: a low level of neurotensin gene message was also detected in the unstimulated PC12 cells after 30 cycles of the PCR. Our findings suggest that oligonucleotides play a role in both quantitative and morphological studies of neuroendocrine expression at the cellular level.

UR - http://www.scopus.com/inward/record.url?scp=0025217554&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025217554&partnerID=8YFLogxK

M3 - Article

AN - SCOPUS:0025217554

VL - 11

SP - 1

EP - 9

JO - Biomedical Research

JF - Biomedical Research

SN - 0388-6107

IS - 1

ER -