Detection of Target Gene Expression in a Bacteria-Infected Mouse Brain

Samenvatting

Source: Zhang, S., et al. Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model. J. Vis. Exp. (2018).

This video demonstrates the detection of target gene expression in a bacteria-infected mouse brain. RNA is extracted, treated with a DNA-degrading enzyme, and reverse-transcribed into cDNA. The cDNA is amplified through thermal cycling with gene-specific primers, while a DNA-binding dye binds to the amplified DNA, emitting fluorescence. The fluorescence signals are analyzed to confirm the presence of the target gene.

Protocol

1. Preparation of Bacteria

Note: Streptococcus suis (S. suis) serotype 2 virulent strain P1/7 was isolated from a diseased pig with meningitis. Strain P1/7 was grown in Todd-Hewitt broth (THB, formula per liter of THB: Heart Infusion, 3.1 g; neopeptone, 20.0 g; dextrose, 2.0 g; sodium chloride, 2.0 g; disodium phosphate, 0.4 g; sodium carbonate, 2.5 g) and plated on Todd-Hewitt agar (THA, formula per liter of THA: Heart Infusion, 3.1 g; neopeptone, 20.0 g; dextrose, 2.0 g; sodium chloride, 2.0 g; disodium phosphate, 0.4 g; sodium carbonate, 2.5 g; agar, 15.0 g) at 37 °C and 5% CO₂.

1. Collection of strain P1/7 p....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Todd Hewitt Broth (THB)	Becton, Dickinson and Company	DF0492078	Dissolve 30 g of the powder in 1 L of purified water. Autoclave at 121° for 15 min.
Agar	DSBIO	16C0050	Dissolve 15 g of the powder in 1 L of THB. Autoclave at 121° for 15 min.
Milli-Q Reference Water Purification System	Merck KGaA	Z00QSVCUS	Without Dnase/ Rnase
NaCl	Tianjin Kemiou Chemical Reagent Co., Ltd	10019318
Na₂HPO₄	Xilong Scientific Co., Ltd	9009012-01-09
KCl	Xilong Scientific Co., Ltd	9009017-01-09
KH₂PO₄	Xilong Scientific Co., Ltd	9009019-01-09
KOH	Xilong Scientific Co., Ltd	9009014-01-09
Glycerol	Sionpharm Chemical Reagent Co., Ltd	10010618	Diluted with equal volume of purified water, autoclave at 121° for 15 min
4% paraformaldehyde	Sionpharm Chemical Reagent Co., Ltd	80096675
25% Glutaraldehyde	Sionpharm Chemical Reagent Co., Ltd	30092436	10-fold diluted with purified water for fixation.
Ethanol	Sionpharm Chemical Reagent Co., Ltd	10009218
Chloroform	Sionpharm Chemical Reagent Co., Ltd	10006818
Spctrophotometre	DeNovix Inc.	DS-11+
Ultrasound cell crusher	NingBo Scientz Biotechnology Co.,Ltd	JY96-IIN
Centrifuge	Hitachi Koki Co., Ltd	CT15RE
Refrigerator	Aucma Co., Ltd	DW-86L500
FastRNA Pro Green Kit	MP Biomedicals	#6045-050
FastPrep-24 Instrument	MP Biomedicals	116005500
Instrument for PCR	SensoQuest GmbH	1124310110
QuantStudio 6 Flex	Thermo Fisher Scientific	4485689
SYBR Premix Ex Taq II	Takara Biomedical Technology (Beijing) Co., Ltd	RR820A
PrimeScript RT reagent kit with gDNA Eraser	Takara Biomedical Technology (Beijing) Co., Ltd	RR047A