Absolute mRNA quantification using the polymerase chain reaction (PCR): a novel approach by a PCR aided transcript titration assay (PATTY)

• Published in: Nucleic acids research Vol. 17; no. 22; pp. 9437 - 9446
• Main Authors: BECKER-ANDRE, M, HAHLBROCK, K
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 25.11.1989
• Subjects: Base Sequence; Biological and medical sciences; Diverse techniques; DNA-Directed DNA Polymerase; Fundamental and applied biological sciences. Psychology; Molecular and cellular biology; Molecular Sequence Data; mRNA; Nucleic Acid Amplification Techniques; Oligonucleotide Probes; Plants - genetics; Polymerase Chain Reaction - methods; RNA - genetics; RNA - isolation & purification; RNA, Messenger - analysis; RNA, Messenger - genetics; Solanum tuberosum; Transcription, Genetic; Polymerase chain reaction; In vitro transcription; Molecular hybridization; Messenger RNA; Complementary DNA; Gel electrophoresis; Site directed mutagenesis; Single stranded DNA; Method; Peptidic synthesis; Nucleic acid; Quantitative analysis
• ISSN: 0305-1048; 1362-4962

The polymerase chain reaction (PCR) is used as part of a new approach to the absolute quantification of mRNA. We describe a PCR aided transcript titration assay (PATTY) which is based on the co-amplification of an in vitro generated transcript differing by a single base exchange from the target mRNA. Identical portions of a total RNA sample are "spiked" with different amounts of this mutated standard RNA, converted to cDNA and amplified by PCR. Because the base exchange creates a novel restriction endonuclease site, the ratio of co-amplified DNA derived from target mRNA to amplified DNA derived from standard RNA can be determined after restriction endonuclease digestion and separation by gel electrophoresis. This method gives accurate results within 24 hours and is useful especially for the quantification of either low-abundance mRNA or more abundant mRNA present in very small amounts of total RNA. The low-abundance mRNA encoding 4-coumarate:CoA ligase (4CL) in cultured potato cells (Solanum tuberosum L.) was measured in a case study. About 100 molecules per assay could be accurately detected by the new method.