Quantitation of mRNA by Polymerase Chain Reaction Nonradioactive PCR Methods

In this laboratory "cook-book", the authors provide a concise guide to PCR-based techniques to quantify nucleic acids in biological and clinical samples using exclusively nonradioactive detection methods, e.g. HPLC, biotin and digoxigenin based protocols. Each method presentation also includes sections on theory, reagents, standards, applicability, limitations, and trouble shooting. In addition to the protocols, the authors also provide the necessary information on: general aspects of nucleic acid quantitation; design of PCR standards; mRNA purification; cDNA synthesis; solution hybridization; DNA sequencing. This laboratory guide enables professionals as well as beginners to adopt easily quantitative PCR protocols into their own clinical or biomedical research.

I Theoretical and Methodical Prerequisites for Using PCR to Quantitate Nucleic Acids.- 1.1 General Aspects and Chances of Nucleic Acid Quantitation by PCR.- 1.2 Design of Suitable Primers and Competitor Fragments for Quantitative PCR.- 1.3 Cloning of Short DNA Fragments and In Vitro Transcription to Generate RNA Standards.- 1.4 Direct Non-lsotopic Sequencing of PCR Products or Standards.- 2Conventional Techniques for mRNA Analysis.- 2.1 Isolation of mRNA.- 2.2 Synthesis of cDNA.- 2.3 Qualitative RT-PCR: Amplification of Synthesized mdr-1 cDNA.- 2.4 Single-Tube RT-PCR.- 2.5 Nonradioactive Determination of PCR Products by Using a DIG-Labeled DNA Probe (Dot Blot).- 2.6 Nonradioactive Northern Blot Hybridization with DIG-Labeled DNA Probes.- 3 Semiquantitative and Quantitative Protocols for Measurement of Nucleic Acids by PCR.- 3.1 Quantitation of mRNA by the ELOSA Technique Using External Standards.- 3.2 Semiquantitative Detection of Viral DNA, e.g. for CMV, by Using the DNA Enzyme Immunoassay (DEIA).- 3.3 HPLC-Analysis of Nucleic Acids.- 3.4 Quantitation of Absolute Numbers of mRNA Copies in a cDNA Sample by Competitive PCR.- Acknowledgment.