Northern blot

PROCEDURE: (1) Extract total RNA or mRNA from tissue/cells. (2) Separate RNA by size using denaturing agarose gel electrophoresis (formaldehyde or similar denaturant to prevent secondary structure). (3) Transfer (blot) RNA from gel to a nylon or nitrocellulose membrane via capillary action. (4) Hybridize membrane with a labeled (radioactive or chemiluminescent) complementary DNA or RNA probe. (5) Wash and visualize by autoradiography or imaging. A band appears where the target RNA is present; band intensity reflects expression level, and band position reflects transcript size.

WHAT IT TELLS YOU: Whether a gene is transcribed in a tissue, relative expression level, transcript size, and alternative splicing (multiple bands/different sizes). Housekeeping genes (e.g., GAPDH, β-actin) serve as loading controls.

BLOT COMPARISON (high-yield): - Southern blot: DNA (detects gene presence, deletions, rearrangements, copy number; e.g., fragile X CGG repeats, restriction fragment length polymorphisms). Named for Edwin Southern. - Northern blot: RNA (gene expression). - Western blot: Protein, using labeled antibodies (e.g., confirmatory HIV test historically, Lyme disease confirmation, prion detection). - Southwestern blot: DNA-binding proteins (detects protein binding to DNA, e.g., transcription factors). - Eastern blot: Post-translational modifications of proteins (less commonly tested). Mnemonic: SNoW DRoP: Southern–DNA, Northern–RNA, Western–Protein.

CLINICAL/BOARD SIGNIFICANCE: Used in research to quantify gene expression; largely replaced in practice by RT-PCR/qPCR and RNA-seq, which are more sensitive, faster, and need less RNA. Remaining advantages: provides transcript size and splice variant information and is relatively straightforward to interpret. Related techniques commonly tested alongside: PCR, microarray (hybridization of cDNA to probes to measure expression of many genes), ELISA (antibody-based protein detection), FISH (fluorescent probe hybridization to chromosomes in situ), and RT-PCR (RNA to cDNA via reverse transcriptase, then amplification).

KEY POINT: The name "Northern" is a play on Southern blot (not a person's name); Southern blot is the only one named after a person.