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High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs
Published on: August 3, 2011
Expression profiling of glycosyltransferases using RT-PCR.
1Department of Pathology, Shinshu University School of Medicine, Matsumoto, Japan.
Methods in Enzymology
|November 23, 2006
Summary
This study details a modified quantitative reverse-transcription polymerase chain reaction (RT-PCR) for accurately measuring low-level messenger RNA (mRNA) in mouse gastric mucosa. The method quantifies glycosyltransferases (GTs) and related genes involved in lymphocyte homing during Helicobacter felis infection.
Area of Science:
- Molecular Biology
- Immunology
- Microbiology
Background:
- Quantitative reverse-transcription polymerase chain reaction (RT-PCR) is crucial for measuring messenger RNA (mRNA) levels.
- Glycosyltransferases (GTs) and related genes are often transcribed at very low levels, posing quantification challenges.
- Understanding gene expression in the context of Helicobacter felis infection and lymphocyte homing is vital.
Purpose of the Study:
- To establish optimized conditions for a quantitative RT-PCR assay.
- To quantify transcript levels of GTs and related genes involved in L-selectin-mediated lymphocyte homing.
- To investigate gene expression in mouse gastric mucosa infected with Helicobacter felis.
Main Methods:
- Modification of conventional RT-PCR protocols.
- Development of a quantitative RT-PCR method without fluorescence-detecting thermal cyclers.
- Application to assaying transcript levels of GTs and related genes.
Main Results:
- Preliminary experiments successfully established conditions for quantitative RT-PCR.
- The method is applicable to low-abundance transcripts like GTs.
- The assay is suitable for accurate analysis of large sample numbers.
Conclusions:
- A modified, accurate, and accessible quantitative RT-PCR method has been developed.
- This method enables precise quantification of low-level mRNA, including GTs.
- The technique is valuable for studying gene expression in complex biological systems like Helicobacter felis-infected gastric mucosa.
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