mRNA for Northern Blotting
Total RNA for northern analysis will frequently result in barely detectable hybridization signals and isolation of mRNA is generally preferred.
Only 1–5% of the total RNA in the cytoplasm of a typical eukaryotic cell is mature mRNA. This mRNA may occur in high, medium, or low abundance. By using mRNA instead of total RNA, cleaner results of greater sensitivity are obtained. The reduced background and increased signal intensity help ensure your results are unambiguous.
- Isolate mRNA from total RNA or directly from crude samples in only 15 minutes.
- The isolation protocol can be scaled up or down.
- Easy and efficient magnetic handling minimizes losses caused by sample manipulations.
- Stringent hybridization and washing buffers along with strong RNase inhibition helps ensure isolation of full-length, intact mRNA.
- Dynabeads® Oligo(dT)25 can be regenerated and reused at least four times.
Isolate mRNA with Dynabeads®
Dynabeads® Oligo(dT)25 allows the isolation of mRNA directly from crude samples or from total RNA. The flexible and scalable method fits any sample size, and is particularly useful for northern analysis starting from small amounts of sample material.
The unique paramagnetic properties of Dynabeads® facilitate easy and efficient handling, whilst also eliminating the requirement for centrifugation, precipitation, and the use of hazardous chemicals. Using a solid-phase enables all procedures to be conducted in a single tube and helps ensure that losses caused by multiple sample manipulations are minimized. This is particularly important when working with small and precious samples or when isolating low-abundance transcripts.
Northern analysis of timecourse expression of Cyclin D1 mRNA in human airway smooth muscle. Samples stimulated with thrombin (T) for 2, 4, 8, or 16 hours to induce expression. C = control without thrombin. Total RNA extracted with TRIzol® reagent. Loaded per lane: 5 μg total RNA (left panel), mRNA extracted from 75 μg total RNA with Dynabeads® Oligo(dT)25 (right panel). Probed with radiolabeled Cyclin D1 and beta-actin and exposed to x-ray film at -20°C overnight (total RNA) or for two days (mRNA). Courtesy of E. Guida and A. Stewart, Bernard O'Brien Institute of Microsurgery, St. Vincents Hospital, VIC, Australia.
- Jakobsen KS et al. Direct mRNA Isolation Using Magnetic Oligo (dT) Beads: A Protocol for All Types of Cell Cultures, Animal and Plant Tissues. Advances in biomagnetic separation. Eaton Publishing 1994;61-71.
- Pines G et al. Cloning and expression of a rat brain L-glutamate transporter. Nature 1992;360:464-467.
- Ainsworth C. Isolation of RNA from Floral Tissue of Rumex acetosa (Sorrel). Plant Mol. Biol. Reporter,1994;12(3):198-203.
- Sæboe-Larssen S and Lambertsson A. A Novel Drosophila Minute Locus Encodes Ribosomal Protein S13. Genetics 1996;143:877-885.
- Hirt H et al. cdc2MsB, a cognate cdc2 gene from alfalfa, complements the G1/S but not the G2/M transition of budding yeast cdc28 mutants. Plant J 1993;4(1):61-69.
- Rovira C and Edström JE. Centromeric polymerase III transcription units in Chironomus pallidivittatus. Nucleic Acids Res. 1996;24(9):1662-1668.
- Theodosiou AM et al."A member of the MAP kinase phosphatase gene family in mouse containing a complex trinucleotide repeat in the coding region. Hum.Mol.Genet. 1996;5(5):675-684.
For Research Use Only. Not for use in diagnostic procedures.