Application of quantitative RT-PCR to the analysis of dopamine receptor mRNA levels in rat striatum

Sheila L. Vrana, Bryan W. Kluttz, Kent E. Vrana

Research output: Contribution to journalArticle

  • 22 Citations

Abstract

A quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) procedure has been developed which selectively amplifies and quantifies the two isoforms of the dopamine D2 receptor. Variability is corrected by the inclusion of a D2 dopamine receptor mRNA standard within each reaction. The internal standard was generated by introducing a point mutation within a D2 cDNA clone that created a unique restriction site within the amplified region. An in vitro transcribed RNA for the internal mutant control is added to the RNA isolated from brain tissue and the mixture is subjected to RT-PCR, digestion with the restriction enzyme, separation of the products by PAGE, and quantification by direct analysis of radioactivity incorporated during the PCR step. The standard is amplified, in the same reaction aas the experimental RNA, using the same primers and RT-PCR conditions. In this manner, the effects of contaminants of the RNA preparation which could affect the amplification procedure are assessed. To insure that the amplification is linear, the number of PCR cycles is minimized. This adaptation avoids 'competitive PCR' and provides for a linear response. Moreover, to obviate non-specific co-amplification, primer annealing steps are performed at or above the melting temperature for the primers, thus increasing signal-to-noise ratios. Finally, primer pairs have been designed which permit amplification of specific fragments for each of the five rat dopamine receptor subtypes. These fragments have unique sizes and so can be differentiated when simultaneously amplified in the same RNA preparations.

LanguageEnglish (US)
Pages127-134
Number of pages8
JournalMolecular Brain Research
Volume34
Issue number1
DOIs
StatePublished - Dec 1 1995

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Dopamine Receptors
Reverse Transcriptase Polymerase Chain Reaction
RNA
Messenger RNA
Dopamine D2 Receptors
Polymerase Chain Reaction
Signal-To-Noise Ratio
Point Mutation
Radioactivity
Freezing
Digestion
Protein Isoforms
Complementary DNA
Clone Cells
Temperature
Brain
Enzymes

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cellular and Molecular Neuroscience

Cite this

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abstract = "A quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) procedure has been developed which selectively amplifies and quantifies the two isoforms of the dopamine D2 receptor. Variability is corrected by the inclusion of a D2 dopamine receptor mRNA standard within each reaction. The internal standard was generated by introducing a point mutation within a D2 cDNA clone that created a unique restriction site within the amplified region. An in vitro transcribed RNA for the internal mutant control is added to the RNA isolated from brain tissue and the mixture is subjected to RT-PCR, digestion with the restriction enzyme, separation of the products by PAGE, and quantification by direct analysis of radioactivity incorporated during the PCR step. The standard is amplified, in the same reaction aas the experimental RNA, using the same primers and RT-PCR conditions. In this manner, the effects of contaminants of the RNA preparation which could affect the amplification procedure are assessed. To insure that the amplification is linear, the number of PCR cycles is minimized. This adaptation avoids \{textquoteleft}competitive PCR\{textquoteleft} and provides for a linear response. Moreover, to obviate non-specific co-amplification, primer annealing steps are performed at or above the melting temperature for the primers, thus increasing signal-to-noise ratios. Finally, primer pairs have been designed which permit amplification of specific fragments for each of the five rat dopamine receptor subtypes. These fragments have unique sizes and so can be differentiated when simultaneously amplified in the same RNA preparations.",
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Application of quantitative RT-PCR to the analysis of dopamine receptor mRNA levels in rat striatum. / Vrana, Sheila L.; Kluttz, Bryan W.; Vrana, Kent E.

In: Molecular Brain Research, Vol. 34, No. 1, 01.12.1995, p. 127-134.

Research output: Contribution to journalArticle

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