Example of real-time quantitative reverse transcription-PCR (Q-RT-PCR) analysis of bacterial gene expression during mammalian infection: Borrelia burgdorferi in mouse tissues.
This unit provides a chronological in-depth description of all protocols needed for quantitative reverse transcription-PCR (Q-RT-PCR) analysis of Borrelia burgdorferi gene expression within infected mouse tissues. Specifically, this unit discusses the extraction of RNA from infected mouse tissues, removal of contaminating genomic DNA from the purified RNA, preparation of cDNA and genomic DNA standards, LightCycler-based PCR, and a relative quantification analysis of the cDNA. Q-RT-PCR as a highly relevant and powerful tool used to detect gene expression by bacteria within mammalian host tissues. It is also an invaluable technique used to measure and assess the small differences in expression that can exist among a set of genes, whereas this type of analysis is not feasible using less-sensitive techniques such as indirect immunofluorescence analysis or qualitative RT-PCR. Although the protocols described herein are tailored for B. burgdorferi, they are broadly applicable to other microbes.
Miller
Jennifer C
JC
University of Kentucky College of Medicine, Lexington, Kentucky, USA.
D000818:Animals; D025065:Borrelia burgdorferi / Q000235:genetics* / Q000378:metabolism; D003264:Containment of Biohazards; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D004586:Electrophoresis; D015964:Gene Expression Regulation, Bacterial; D008193:Lyme Disease / Q000382:microbiology*; D051379:Mice; D012329:RNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013053:Spectrophotometry
D004269:DNA, Bacterial; D018076:DNA, Complementary; D012329:RNA, Bacterial