Digital image analysis for rapid quantification of total RNA and cDNA for SMART (TM)-PCR

摘要

Many molecular biology techniques such as cDNA library construction, differential display, subtractive or northern hybridization, microarrays, and direct cDNA selection need a high quantity of initial mRNA or cDNA, typically several micrograms. In many instances when experimental material is very limited or unique, a researcher does not have enough starting RNA to implement these powerful approaches. Fortunately, the development of SMARTTM (switching mechanism at 5' end of RNA transcript) technology (Clontech Laboratories, Palo Alto, CA, USA) and the availability of long-distance (LD) PCR allow one to overcome this problem. SMART technology produces ss-cDNA enriched for complete 5' untranslated regions (UTRs) of mR-NAs. Full-length cDNA molecules contain incorporated sequences for two primers and can be amplified exponentially in the following LD-PCR. Truncated molecules contain sequence only for one primer and can only be amplified linearly. Consequently, after 15-25 cycles of LD-PCR, it is possible to receive several micrograms of full-length ds-cDNA, starting only with 50-1000 ng total RNA (1) and maintaining at the same time the original message profile (2,3). The most important step in this approach, which dramatically influences the final result, is the condition of the ds-cDNA amplification (number of PCR cycles and longevity of different PCR steps). To obtain the best results, PCR should be in the exponential phase of the reaction.