mTRAP™ Total

The mTRAP Total Kit was created for single-pass enrichment of mRNA from total RNA. Reagents and plasticware are included for 12 mRNA isolations. Each reaction can isolate mRNA from up to 500 µg total RNA and will yield 10-15 µg mRNA.

Figure 1: Flowchart of the procedure for the mTRAP Maxi, Midi and Total Kits.

Figure 2: Pure mRNA from total RNA in one passage.
mRNA was isolated from 500 µg of total RNA in duplicate, quantified by spectrophotometry and run on a 0.8% agarose gel.
Lane 1: 1 kb DNA Ladder
Lanes 2-3: 2 µg mRNA isolated in one pass with the mTRAP Total Kit.

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 DNA^1-4. 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 PNAs^5-7, which have a number of properties that make them ideal for mRNA isolation.

PNA molecules bind to complementary nucleic acids with a much higher affinity than 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 75°C 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: Higher yields of pure mRNA.
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 µg 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.

Lane 1: 1 kb DNA Ladder
Lane 2: mRNA isolated using Company A’s kit
Lane 3: mRNA isolated using Company Q’s kit
Lane 4: mRNA isolated using mTRAP™ Maxi Kit
Lane 5: mRNA isolated using Company I’s kit

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

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.

Contents & Storage

Poly T gripNA™ Probe, Streptavidin Beads, 2X Binding and Wash Buffers, DEPC and HPLC Water, Microcentrifuge Tubes and TouchDown™ Precipitation Reagent. Storage conditions vary from room temperature to -20°C, see manual for details. All reagents are guaranteed stable for 6 months when stored properly.