Table 1 Summary of the modifications made for the conventional Northern blotting technique and advantages resulted
From: Modified Northern blot protocol for easy detection of mRNAs in total RNA using radiolabeled probes
Steps | Conventional Northern blotting | Modifications | Advantages |
|---|---|---|---|
RNA denaturation before electrophoresis | No EtBr in RNA samples | Heating RNA samples in the presence of EtBr before loading the gel | Possible to verify the integrity of RNA and evaluate the quality of the size-separation at any time even during electrophoresis, to check the transfer efficiency immediately after blotting |
Formaldehyde concentration in agarose gels | Great variation in different protocols, much higher (e.g., 18%) or even much lower to 3% | A moderate concentration of 12% | Achieved the best combination of denaturation to both RNA and RNases during electrophoresis and no hindrance to subsequent blotting |
Capillary transfer | Only transfer setup | An accessory setup along the both sides of the transfer setup | Avoidance the collapse of the setup and ensured the efficient and successful transfer |
Hybridization | In hybridization oven | In a staining jar placed into an enamel square tray with a lid | Greatly decreased cost while still efficiently protection of investigators from radioactivity and proceeding of the hybridization |
Post-hybridization washes | Followed by a blocking step of under high and low stringency sequentially for scheduled time | Under only moderate-stringency till the level of radioactivity retained on the filter decreased to 20~50 cps | Maximally retaining the specific hybridized probes on the filter thus improved the detection sensitivity; Possible to monitor the progress of posthybridization detection |