1/14/2024 0 Comments Northern blot analysis of rna![]() After exposure, wash the membrane in TBST for 10 min. Take the image on high-resolution mode from 1 s exposure up to 3 min exposure with appropriate intervals. Expose the membrane with your imaging system.Incubate the membrane with ECL substrate for 5 min.Wash the membrane four times for 10 min each in 10 mL of TBST with gentle shaking. Flip the membrane so it is RNA-side up.Incubate the membrane with appropriate secondary antibody (goat anti-rabbit IgG-HRP (1:20,000 dilution, ab97051) or anti-mouse IgG-HRP) in blocking buffer for 1 h at room temperature with gentle shaking. Flip the membrane so it is RNA-side down.Wash the membrane three times for 10 min each in 10 mL of TBST with gentle shaking. Expression profiling of single genes or small gene panels - Northern blot - Ribonuclease protection assay - In-situ hybridization - Real-time reverse. Discard the blocking buffer, incubate the membrane with primary antibody (at given dilution, usually 1 μg/mL working IgG) in 10 ml blocking buffer overnight at +4☌ with gentle shaking.Incubate the membrane (RNA-side down to prevent accidental drying out of the membrane) in 10 ml blocking buffer for 1 h at room temperature with gentle shaking.Wash the membrane in 10 mL of TBST (1X TBS, 0.1% Tween-20), for 5 min at room temperature with gentle shaking to wash off the unbound RNA.Crosslink RNA to the membrane with UV light: 125 mJoule/cm2 at 254 nM. Remove the dish lid and ensure the membrane is RNA side up. Transfer the dish with membrane immediately into the chamber of SG Linker (with 254 nM bulb).Change tips after each loading, even between the same sample. Shortly after southern blotting was developed, the premise of this analytical technique was applied to the measurement of the size and amount of RNA transcripts. Let pipetted RNA droplet diffuse onto the membrane via surface tension. NB Avoid touching the membrane with the pipette tip. ![]()
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