( 0.04 ...)High-Throughput Isolation of Total RNA

RNeasy 96

1.0 0.22

16 2.3

1.9 0.05

RNeasy 96 + DNase

0.6 0.13

8 1.9

1.9 0.07

RT-PCR was performed on RNA samples isolated with both kits using primers specific for GAPDH. These primers amplify a 209-bp product from cDNA and from pseudogene sequences in genomic DNA. We performed cDNA synthesis reactions both in the presence and absence of reverse transcriptase, followed by PCR as an assay for DNA contamination. No DNA was detected in the StrataPrep 96 kit samples using either the standard or the alternate protocol, whereas both of the RNeasy 96 samples demonstrated DNA contamination, even when DNase treatment was included (Figure 5).

Fig.5

Conclusions

The StrataPrep 96 total RNA purification kit is the method of choice for isolating RNA from as many as 96 cultured cell samples. The method generates pure, intact RNA with high yields. While amplification by conventional and molecular beacon RT-PCR can be achieved for samples isolated from as few as 10 cells, the StrataPrep 96-well RNA kit is clearly superior when compared with the RNeasy kit because Stratagenes kit yields RNA at a higher concentration with no detectable DNA contamination.

Acknowledgments

The authors thank Gothami Padmabandu and Reinhold Mueller for performing molecular beacon RT-PCR.

REFERENCES

1. Sambrook, J., et al. (1989) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Labo
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