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

作者: Michael Becker-André , Klaus Hahlbrock

DOI: 10.1093/NAR/17.22.9437

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

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