This study outlines a method for analyzing infection-responsive gene expression in mouse brain tissue following bacterial infection. The protocol includes RNA extraction, cDNA synthesis, and quantitative real-time PCR to assess gene expression levels.
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 brain tissue into five tubes. Take up to 100 milligrams of brain tissue and add 1 milliliter of lysis solution to each tube containing the lysis matrix provided by the kit.
Extract the RNA following the steps listed in the text protocol. Next, perform cDNA synthesis, including elimination of genomic DNA, using a thermocycler with a reverse transcription reagent kit. Add 1 microgram of RNA to a tube containing 2 microliters of 5x genomic DNA elimination buffer and 1 microliter of genomic DNA elimination enzyme.
Add RNase-free water until the volume reaches 10 microliters. Incubate for 2 minutes at 42 degrees Celsius. Add a 10 microliters of master mix to the reaction solution and then mix gently.
Proceed immediately with the reverse transcription reaction by placing the tube at 37 degrees Celsius for 15 minutes. Then transfer the reaction to 85 degrees Celsius for 5 seconds. Perform the quantitative real-time PCR analysis using a real-time PCR machine with a cyber quantitative real-time PCR kit. Following the kit instructions, combine the enzyme, primers, 50x ROX reference dye two, and cDNA template. Then add RNase-free water until a total volume of 20 microliters is reached.
Run each sample in triplicate using the thermal parameters listed in the text protocol and calculate the relative fold-change based on the two minus delta delta CT method.
繁體中文
