Characterization of RNA Using Continuous RT-PCR Coupled with ELOSA

Abstract

Characterization of RNA using Northern blotting or RNase protection assay is achieved by a two step procedure including a separation step and a detection step. Specificity and sensitivity are conferred by both the separation process and hybridtzation with radiolabeled probes. In both techniques, the observed results are validated after normalization of RNA performed by using a probe generally targeting a housekeeping gene. The greatest sensitivity was reached with the recently introduced reverse transcription-polymerase chain reaction (RT-PCR). However, because of the sensitivity of RT-PCR, false positives could present a problem. The selection of primer sets overlapping splice Junctions could prevent amplification of potentially contaminating DNA in the RNA preparation. Using an RT-PCR process that does not require the reopening of the test tube could reduce the risk of introducing contaminating cloned cDNA or RNA during handling. False negatives could also present a problem. Quality of the RNA preparation (and quantity) could be checked by amplification of a housekeeping gene that informs one of the presence of potentially contaminating inhibtiors of the enzymatic reaction. The specrficrty in RT-PCR assays is generally achieved using a detection analysis by Southern blot, dot blot, or restrrctron mapping. These techniques are generally used in specialized laboratories and could be replaced by microtiter plate or tube format assays well-adapted to clmical applications (1). On such a format assay, PCR products must be captured on a solid phase and further detected by direct or indirect nonisotopic labeling (reviewed in ref. 2).