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mRNA ant Total RNA Isolation

mTRAP

isolate more mRNA with gripNAs

Why mTRAP and gripNAs?

• Isolates substantially more mRNA than oligo dT
• Reduces ribosomal RNA and genomic DNA contamination
• Captures mRNA with secondary structure
• Purifies a more representative mRNA population
• Includes TouchDown for precipitating small quantities of nucleic acids

mTRAP Kits isolate large yields of high-quality mRNA from mammalian cells, tissue and total RNA. mTRAP Maxi, Midi and 96 Kits optimize isolation of mRNA from different sample sizes, while mTRAP Total is intended for isolation of mRNA from total RNA. All mTRAP Kits utilize Active Motifs Poly T gripNA Probe*, which has a higher affinity and specificity for mRNA than oligo dT. The result is more mRNA per sample, with much less ribosomal RNA and genomic DNA contamination.

The mTRAP Advantage

Peptide Nucleic Acids (PNAs) are DNA analogs in which the nucleotides are attached to an N-(2-aminoethyl)glycine backbone instead of to deoxyribose, as in DNA1-4. However, poor water solubility and a tendency to self-aggregate have limited the utility of traditional PNAs. Active Motif has overcome these shortcomings by developing negatively charged PNAs5-7. This class of PNAs has a number of properties that make them ideal for mRNA isolation.

PNA molecules bind to complementary nucleic acids with a much higher affinity as compared to oligo dT. This enables the use of binding and wash buffers that contain significantly lower salt concentrations than conventional methods. These conditions effectively eliminate stem/loop structures, enabling the Poly T gripNA Probe to hybridize to the poly A tails on all mRNA molecules. The low-ionic conditions also remove contaminating nucleic acid and protein molecules that may have formed weak, non-specific interactions with the Poly T gripNA Probe. Consequently, mTRAP Kits provide higher yields of a more representative population of mRNA, with significantly reduced ribosomal RNA and protein contaminations (Figure 1).

Because genomic DNA can contain stretches of poly As, co-isolation of genomic DNA is frequently a problem when isolating mRNA with oligo dT. A useful characteristic of the Poly T gripNA Probe is that it binds to complementary DNA with a higher affinity than it does to complementary RNA. This enables the mRNA to be selectively eluted off the Poly T gripNA Probe. Even during the 75C elution, genomic DNA remains bound. For absolute certainty that no contaminating genomic DNA is co-isolated, an optional DNase step can be performed during isolation because PNAs are resistant to enzymatic degradation. For complete information on the benefits of PNAs, please download one of the mTRAP manuals.

Figure 1:
mRNA was isolated from 1 and 2 x 10⁸ HeLa cells using mTRAP Maxi and three other suppliers kits. Eluted mRNA was quantified by spectrophotometer and plotted. 2 g of mRNA from each 1 x 10⁸ sample was run on a 0.8% agarose gel. mTRAP-isolated mRNA shows no genomic DNA and less rRNA.

References

1. Nielsen P.E., et al. (1991) Science 254: 1497-1500.
2. Egholm M, et al. (1992) J. Am. Chem. Soc. 114: 1895-1897.
3. Harvey JC et al. (1992) Science 258: 1481-1485.
4. Buchardt O, et al. (1993) Trends Biotechnol. 11: 384-386.
5. Efimov VA, et al. (1998) NAR 26: 566-575.
6. van der Laan, et al. (1996) Tetrahedron Lett. 37: 7857-7860.
7. Efimov VA, et al. (1999) NAR 27: 4416-4426.

* Patent pending