JoVE Encyclopedia of Experiments
Microbiology
0 views • 4:02 min • February 2nd, 2026
Take total RNA extracted from a mouse brain infected with pathogenic bacteria. The extract contains transcripts from infection-responsive genes.
Add a DNA-degrading enzyme and incubate to degrade residual genomic DNA.
Add a master mix containing reverse transcriptase (RT) enzyme, nucleotides, and nonspecific primers.
Incubate to synthesize complementary DNA (cDNA).
Introduce a high temperature to inactivate the RT enzyme.
Take a master mix containing DNA polymerase, nucleotides, a double-stranded DNA-binding dye, and primers specific to a target gene.
Add the cDNA and apply heat to denature the DNA.
The primer binds to the target cDNA, enabling new DNA strand synthesis.
The cycle is repeated to amplify the target gene.
The dye binds to the amplified DNA and emits fluorescence, which is recorded at the end of each cycle.
Increase in fluorescence confirm the expression of the infection-responsive gene in the infected mouse brain.
Infect all mice using planktonic cells or biofilm state cells and perform euthanasia as described in the text protocol. Next, extract the total RNA from brain tissue using an RNA extraction kit.
For each mouse, use whole brain tissue for extracting RNA. Divide whole bra
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