Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a<i>P</i>CR aided<i>t</i>ranscipt<i>t</i>itration assay (PATTY)

TLDR

PCR is employed in a novel approach for absolute mRNA quantification. The study introduces PATTY, a PCR‑aided transcript titration assay that co‑amplifies a single‑base‑mutated standard with target mRNA. PATTY spiked total RNA with varying amounts of a single‑base‑mutated standard, converted it to cDNA, amplified by PCR, and quantified the target/standard ratio via restriction digestion and gel electrophoresis. The assay delivers accurate results within 24 h, detecting as few as ~100 molecules, and was successfully applied to quantify low‑abundance 4CL mRNA in potato cells.

Abstract

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 amount of this mutated standard RNA, converted to cDNA and amplified by PCR. Because the base exchane 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 seperation by gel electrophoresis. This method gives accurate results within 24 hours and is useful especially for the quantification of either lowabundance 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 be accurately detected by the new method.