Direct quantitation of RNA transcripts by competitive single-tube RT- PCR and capillary electrophoresis

N. D. Borson, M. A. Strausbauch, P. J. Wettstein, R. P. Oda, S. L. Johnston, J. P. Landers

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

Attempts are frequently made to semiquantitate mRNA as a means of circumventing the laborious and time-consuming process of quantitation that is inherent in the use of competitor templates. However, semiquantitative approaches present the risk of generating non-reproducible data due to tube- to-tube variability and/or misinterpretation of quantities of product being generated during the plateau phase of PCR. Subsequently, it is difficult to compare semiquantitative data from separate experiments, and comparisons of levels of mRNA transcript from genes that amplify with different primer pairs cannot be made. Thus, reliable methods for mRNA quantitation continue to rely on the use of internal standardization. In this report, we describe a strategy for dependable quantitation of low-abundance mRNA transcripts based on quantitative competitive reverse transcription PCR (QC-RT-PCR) coupled to capillary electrophoresis (CE) for rapid separation and detection of products. Recommendations are included for the design of RNA competitors that can be paired with target RNA for cDNA synthesis primed with a gene-specific primer; these synthesized cDNAs are then co-amplified directly in the same tube using a single primer pair. We describe (i) a protocol for a single- tube RT-PCR that provides for cDNA synthesis and subsequent PCR amplification of target and competitor in identical reaction environments at each critical enzymatic step, (ii) a unique hot-start provision for optimizing precise and consistent PCR amplifications and (iii) a method for rapid PCR product separation, detection and quantitation by CE and laser-induced fluorescence.

Original languageEnglish (US)
Pages (from-to)130-137
Number of pages8
JournalBioTechniques
Volume25
Issue number1
StatePublished - 1998

Fingerprint

Capillary electrophoresis
Capillary Electrophoresis
RNA
Polymerase Chain Reaction
Messenger RNA
Complementary DNA
Amplification
Genes
Transcription
Standardization
Fluorescence
Reverse Transcription
Lasers
Experiments

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Clinical Biochemistry

Cite this

Borson, N. D., Strausbauch, M. A., Wettstein, P. J., Oda, R. P., Johnston, S. L., & Landers, J. P. (1998). Direct quantitation of RNA transcripts by competitive single-tube RT- PCR and capillary electrophoresis. BioTechniques, 25(1), 130-137.

Direct quantitation of RNA transcripts by competitive single-tube RT- PCR and capillary electrophoresis. / Borson, N. D.; Strausbauch, M. A.; Wettstein, P. J.; Oda, R. P.; Johnston, S. L.; Landers, J. P.

In: BioTechniques, Vol. 25, No. 1, 1998, p. 130-137.

Research output: Contribution to journalArticle

Borson, ND, Strausbauch, MA, Wettstein, PJ, Oda, RP, Johnston, SL & Landers, JP 1998, 'Direct quantitation of RNA transcripts by competitive single-tube RT- PCR and capillary electrophoresis', BioTechniques, vol. 25, no. 1, pp. 130-137.
Borson ND, Strausbauch MA, Wettstein PJ, Oda RP, Johnston SL, Landers JP. Direct quantitation of RNA transcripts by competitive single-tube RT- PCR and capillary electrophoresis. BioTechniques. 1998;25(1):130-137.
Borson, N. D. ; Strausbauch, M. A. ; Wettstein, P. J. ; Oda, R. P. ; Johnston, S. L. ; Landers, J. P. / Direct quantitation of RNA transcripts by competitive single-tube RT- PCR and capillary electrophoresis. In: BioTechniques. 1998 ; Vol. 25, No. 1. pp. 130-137.
@article{def0118249f54d6988ec6889ec690c59,
title = "Direct quantitation of RNA transcripts by competitive single-tube RT- PCR and capillary electrophoresis",
abstract = "Attempts are frequently made to semiquantitate mRNA as a means of circumventing the laborious and time-consuming process of quantitation that is inherent in the use of competitor templates. However, semiquantitative approaches present the risk of generating non-reproducible data due to tube- to-tube variability and/or misinterpretation of quantities of product being generated during the plateau phase of PCR. Subsequently, it is difficult to compare semiquantitative data from separate experiments, and comparisons of levels of mRNA transcript from genes that amplify with different primer pairs cannot be made. Thus, reliable methods for mRNA quantitation continue to rely on the use of internal standardization. In this report, we describe a strategy for dependable quantitation of low-abundance mRNA transcripts based on quantitative competitive reverse transcription PCR (QC-RT-PCR) coupled to capillary electrophoresis (CE) for rapid separation and detection of products. Recommendations are included for the design of RNA competitors that can be paired with target RNA for cDNA synthesis primed with a gene-specific primer; these synthesized cDNAs are then co-amplified directly in the same tube using a single primer pair. We describe (i) a protocol for a single- tube RT-PCR that provides for cDNA synthesis and subsequent PCR amplification of target and competitor in identical reaction environments at each critical enzymatic step, (ii) a unique hot-start provision for optimizing precise and consistent PCR amplifications and (iii) a method for rapid PCR product separation, detection and quantitation by CE and laser-induced fluorescence.",
author = "Borson, {N. D.} and Strausbauch, {M. A.} and Wettstein, {P. J.} and Oda, {R. P.} and Johnston, {S. L.} and Landers, {J. P.}",
year = "1998",
language = "English (US)",
volume = "25",
pages = "130--137",
journal = "BioTechniques",
issn = "0736-6205",
publisher = "Eaton Publishing Company",
number = "1",

}

TY - JOUR

T1 - Direct quantitation of RNA transcripts by competitive single-tube RT- PCR and capillary electrophoresis

AU - Borson, N. D.

AU - Strausbauch, M. A.

AU - Wettstein, P. J.

AU - Oda, R. P.

AU - Johnston, S. L.

AU - Landers, J. P.

PY - 1998

Y1 - 1998

N2 - Attempts are frequently made to semiquantitate mRNA as a means of circumventing the laborious and time-consuming process of quantitation that is inherent in the use of competitor templates. However, semiquantitative approaches present the risk of generating non-reproducible data due to tube- to-tube variability and/or misinterpretation of quantities of product being generated during the plateau phase of PCR. Subsequently, it is difficult to compare semiquantitative data from separate experiments, and comparisons of levels of mRNA transcript from genes that amplify with different primer pairs cannot be made. Thus, reliable methods for mRNA quantitation continue to rely on the use of internal standardization. In this report, we describe a strategy for dependable quantitation of low-abundance mRNA transcripts based on quantitative competitive reverse transcription PCR (QC-RT-PCR) coupled to capillary electrophoresis (CE) for rapid separation and detection of products. Recommendations are included for the design of RNA competitors that can be paired with target RNA for cDNA synthesis primed with a gene-specific primer; these synthesized cDNAs are then co-amplified directly in the same tube using a single primer pair. We describe (i) a protocol for a single- tube RT-PCR that provides for cDNA synthesis and subsequent PCR amplification of target and competitor in identical reaction environments at each critical enzymatic step, (ii) a unique hot-start provision for optimizing precise and consistent PCR amplifications and (iii) a method for rapid PCR product separation, detection and quantitation by CE and laser-induced fluorescence.

AB - Attempts are frequently made to semiquantitate mRNA as a means of circumventing the laborious and time-consuming process of quantitation that is inherent in the use of competitor templates. However, semiquantitative approaches present the risk of generating non-reproducible data due to tube- to-tube variability and/or misinterpretation of quantities of product being generated during the plateau phase of PCR. Subsequently, it is difficult to compare semiquantitative data from separate experiments, and comparisons of levels of mRNA transcript from genes that amplify with different primer pairs cannot be made. Thus, reliable methods for mRNA quantitation continue to rely on the use of internal standardization. In this report, we describe a strategy for dependable quantitation of low-abundance mRNA transcripts based on quantitative competitive reverse transcription PCR (QC-RT-PCR) coupled to capillary electrophoresis (CE) for rapid separation and detection of products. Recommendations are included for the design of RNA competitors that can be paired with target RNA for cDNA synthesis primed with a gene-specific primer; these synthesized cDNAs are then co-amplified directly in the same tube using a single primer pair. We describe (i) a protocol for a single- tube RT-PCR that provides for cDNA synthesis and subsequent PCR amplification of target and competitor in identical reaction environments at each critical enzymatic step, (ii) a unique hot-start provision for optimizing precise and consistent PCR amplifications and (iii) a method for rapid PCR product separation, detection and quantitation by CE and laser-induced fluorescence.

UR - http://www.scopus.com/inward/record.url?scp=0031847760&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031847760&partnerID=8YFLogxK

M3 - Article

C2 - 9668987

AN - SCOPUS:0031847760

VL - 25

SP - 130

EP - 137

JO - BioTechniques

JF - BioTechniques

SN - 0736-6205

IS - 1

ER -