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.
Steps for northern blot analysis include RNA isolation, probe generation, denaturing samples, transferring to a charged nylon membrane, and immobilized for hybridization, followed by washing and detection. Learn more with our Step-by-Step Guide to Northern Blot Analysis.
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 time-course 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.
For Research Use Only. Not for use in diagnostic procedures.