( 07TURBO DNA- free to rem...)Efficient Cellular Fractionation for RNA ,,, and Protein Isolation

07">TURBO DNA-free to remove trace contamination of genomic DNA and analyzed by end-point RT-PCR with the RETROscript Kit (Ambion) and pairs of primers specific for the fibronectin gene (Figure 2). Because the PCR was performed under non-quantitative conditions (35 cycles), both forms of the alternatively spliced fibronectin exon EIIIb mRNA were detected in total, cytoplasmic, and nuclear fractions at similar levels. In contrast, no unspliced fibronectin pre-mRNA was present in the cytoplasmic fraction showing efficient cellular fractionation. This result confirms that the PARIS fractionation procedure is ideal for various applications, from studying protein or RNA localization to preparing full-length cDNA libraries free of contaminating nuclear intronic sequences.

Figure 2. Analysis of Fibronectin pre-mRNA Splicing. The mutually exclusive alternative splicing of fibronectin exon EIIIb was analyzed by RT-PCR (35 cycles) using total (T), cytoplasmic (C), and nuclear (N) RNA isolated from 293 cells with the PARIS Kit. Using an intron-specific primer, the unspliced form was detected only in the total and nuclear fractions. No PCR products were detected in the minus RT and minus RNA controls (data not shown).

Isolation of Native Protein
Unlike other RNA and protein isolation reagents, the PARIS procedure does not require a phenol/chloroform extraction followed by tedious precipitation and re-suspension steps. Furthermore, native proteins are recovered in buffers that are compatible with various downstream analysis. This means that protein fractions can be used not on
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