The Northern blotting procedure is straightforward and provides opportunities to evaluate progress at various points (e.g., intactness of the RNA sample and how efficiently it has transferred to the membrane). It can also be used to directly compare the relative abundance of a given message between all the samples on a blot. The most compelling of these is that it is the easiest method for determining transcript size, and for identifying alternatively spliced transcripts and multigene family members. Northern analysis presents several advantages over the other techniques. Northern analysis remains the standard for detection and quantitation of mRNA levels despite the advent of more sensitive techniques. In situ hybridization is used to localize expression of a particular gene within a tissue or cell type, and RT-PCR is the most sensitive method for detecting and quantitating gene expression. (See chart.) In general, Northern analysis is the only method that provides information about transcript size, whereas NPAs are the easiest way to simultaneously examine multiple messages (Figure 1). However, the methods each have inherent advantages and/or limitations. In theory, each of these techniques can be used to detect specific RNAs and to precisely determine their expression level. Here, we review four popular methods: Northern blot analysis, nuclease protection assays (NPA), in situ hybridization, and reverse transcription-polymerase chain reaction (RT-PCR). A number of widely used procedures exist for detecting and determining the abundance of a particular mRNA in a total or poly(A) RNA sample. Add 100µg/mL denatured salmon sperm DNA (or Herring testes DNA) just before use.Molecular characterization of any gene usually includes a thorough analysis of the temporal and spatial distribution of RNA expression.Perform autoradiography making sure to mark the orientation of the membrane on the film.įormamide Prehybridization/Hybridization Solution.Do not allow membrane to dry out if it is to be stripped and reprobed. Blot excess liquid and cover in UV-transparent (SaranWrap) plastic wrap. Remove final wash and rinse membrane in 2X SSC at room temperature.High stringency wash (optional): Change wash solution with prewarmed (68☌) 0.2X SSC/0.1% SDS and incubate with rotation at 68☌ for 15 minutes.Medium stringency wash (optional): Change wash solution with prewarmed (42☌) 0.2X SSC/0.1% SDS and incubate with rotation at 42☌ for 15 minutes.Low stringency wash: Replace wash solution with 0.2X SSC/0.1% SDS and incubate for 5 minutes with rotation at room temperature.Pour wash solution into radioactive waste, replace wash solution and repeat. Incubate at room temperature for 5 minutes.If using resealable bags, membrane can be transferred to a plastic dish for the following washes. Pour off the hybridization solution into the radioactive waste, and replace with at least equal volume of 2X SSC/0.1% SDS.* The probe concentration in the hybridization solution should be 10 ng/mL if the specific activity is 10 8 dpm/µg or 2 ng/mL if the specific activity is 10 9 dpm/µg.

The bag must then be carefully be resealed to avoid leaking radioactivity. If using resealable bags the probe can be added by inserting a syringe into an uncut corner and injection of the probe. Pipet the desired volume * of probe into the hybridization tube and continue to incubate with rotation overnight at 42☌ (DNA-probe) or 60☌ (RNA-probe).Denatured probe should be added to the hybridization solution as soon as possible after denaturation. If the probe is dsDNA, denature by boiling for 5 minutes, and immediately place it on ice.

If using resealable bags, it can be shaken or rocked slowly in a suitable incubator or water bath.

Place the tube in a hybridization oven and incubate with rotation 15 minutes (3 hours if using a nitrocellulose membrane) at 42☌ (DNA-probe) or 60☌ (RNA-probe).

Then a corner of the bag can be cut off to allow pipetting of the prehybridization/hybridization solution into the bag. In which case, the membrane should be sealed into a bag after being wetted. Alternatively one can use resealable bags.

Place the membrane in a hybridization tube with the RNA-side facing in, and add ~1mL formamide prehybridization/hybridization solution per 10 cm 2.

Wet the membrane carrying the immobilized RNA in 6X SSC.

If the membrane was blotted from a glyoxal gel, soak the membrane in 20 mM Tris-HCl (pH 8.0) for 5 minutes to disassociate the glyoxal from the bound RNA.

formamide prehybridization/hybridization solution.

Membrane (nylon or nitrocellulose) with immobilized RNA.

The hybridization step of a northern blot allows identification of RNA blotted and immobilized on a membrane with a radioactive (or otherwise labeled) nucleic acid probe. Adapted from Current Protocols in Molecular Biology

Northern hybridization