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Detection of properdin mRNA in human peripheral blood monocytes and spleen
1Iowa City Veterans Administration Medical Center, IA.
Abstract:
Properdin serves a critical role in the alternative pathway of complement by stabilizing the C3bBb complex. Early studies to determine the properdin sequence relied on amino acid sequencing of enzymatically cleaved properdin and only yielded partial sequence data. Recently Nolan et al. reported a properdin mRNA sequence obtained from the U937 myelomonocytic cell line. We sought to detect properdin mRNA in two normal human tissues and to compare those sequences with that obtained from the U937 cell line. Cytoplasmic RNA harvested from human spleen and peripheral blood monocytes served as a template for first strand synthesis. The cDNA was then used as a template for polymerase chain reaction. A properdin message was detected in both spleen and peripheral blood monocytes but not in peripheral blood neutrophils. The sequence was nearly identical to that obtained from the U937 cell line. These experiments demonstrate that peripheral blood may be used as a ready source for properdin mRNA and will faster studies to define the defect in properdin-deficient patients.
Insights
Properdin mRNA is detectable in human spleen and peripheral blood monocytes, not neutrophils. This finding facilitates research into properdin deficiency by utilizing peripheral blood as a readily available source of properdin mRNA.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Properdin is crucial for the alternative complement pathway, stabilizing the C3bBb complex.
- Previous properdin sequencing efforts yielded incomplete data.
- A recent study reported properdin mRNA sequence from the U937 cell line.
Purpose of the Study:
- To detect properdin mRNA in normal human spleen and peripheral blood monocytes.
- To compare these sequences with the previously reported U937 cell line sequence.
Main Methods:
- Cytoplasmic RNA was isolated from human spleen and peripheral blood monocytes.
- First-strand cDNA synthesis was performed using the harvested RNA.
- Polymerase chain reaction (PCR) was employed to amplify properdin cDNA.
Main Results:
- Properdin mRNA was detected in both spleen and peripheral blood monocytes.
- Properdin mRNA was not detected in peripheral blood neutrophils.
- The detected sequences were highly similar to the U937 cell line properdin mRNA.
Conclusions:
- Peripheral blood monocytes are a viable source for properdin mRNA detection.
- This study simplifies obtaining properdin mRNA for future research.
- Facilitates investigations into the genetic defects in properdin-deficient patients.