Practice question

Northern blotting is historically named by analogy to Southern blotting and is dedicated to RNA target analysis. Total cellular RNA or poly(A) enriched mRNA is denatured with formaldehyde or glyoxal to eliminate secondary structures and separated on agarose gel according to length. After capillary or electro-transfer to positively charged nylon membrane and UV crosslinking, transcripts are hybridized with labeled antisense DNA or RNA probes. Autoradiographic signal indicates transcript size and abundance. Although largely supplanted by quantitative RT-PCR and RNA sequencing for sensitivity, Northern remains gold standard for validating transcript integrity and isoform size without amplification bias.