Differential-Display Reverse Transcription-PCR (Ddrt-Pcr)

ISBN-13: 9783540632979 / Angielski / Miękka / 1998 / 124 str.

B. Maresca; Sergio Colonna-Romano; A. Leone

Opis

This manual describes the technique of differential-display reverse transcription-PCR. It contains step-by-step instructions, practical hints and experimental strategies for every protocol.

Spis treści

Abbreviations.-Overview.-Preparation of Total RNA.-Differential Display.-Size Separation of cDNA Fragments.-Isolation of Differentially Expressed cDNA Fragments.-Reamplification of Eluted cDNA.-Cloning of Amplified cDNA Fragments.-Checking Subcloned Fragments.-Confirmation of Differential Expression of Cloned cDNA Fragments.-Northern Blot Affinity Capturing of cDNA.-Sequencing of Differentially Expressed cDNA Fragments.-References.-Appendix: Materials, Equipment, Reagents, and Suppliers.-Subject Index.

Blurb

Identification of differentially expressed genes is one of the major challenges in molecular biology. Several techniques allow the cloning of such sequences. However, methods such as RNA subtraction or differential hybridization are time-consuming and require large amounts of mRNA.
Recently, a new approach has successfully been developed: Differential-Display Reverse Transcription-PCR (DDRT-PCR). This technique has been proven to be highly effective in identifying sequences that are differentially expressed in various cell types. The most striking advantage is, however, that only nanograms of total RNA are sufficient. Thus every mRNA species expressed in the cell system can be investigated, even those present at very low levels.