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Related Experiment Videos

RT-PCR from eosinophil-depleted leukocytes without RNA extraction: cell selection using streptavidin PCR tubes.

M M Hämäläinen1, J U Eskola, J Hellman

  • 1Department of Clinical Chemistry, Turku University Central Hospital, FIN-20520, Turku, Finland. mauham@utu.fi

Clinical Biochemistry
|February 13, 2001
PubMed
Summary

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This study developed a novel method using streptavidin-coated PCR tubes to remove eosinophils from leukocytes, enabling RNA extraction-free RT-PCR. This technique improves reproducibility for molecular diagnostics.

Area of Science:

  • Molecular Biology
  • Immunology
  • Biotechnology

Background:

  • Eosinophil-derived neurotoxin (EDN) is a major RNase activity in leukocytes.
  • Removing eosinophils allows for RT-PCR analysis from 10^5 leukocytes without RNA extraction.

Purpose of the Study:

  • To develop a method for selecting eosinophil-free leukocytes for RT-PCR analysis.
  • To utilize streptavidin-coated PCR tube strips for this selection process.

Main Methods:

  • Polypropylene PCR tubes were coated with streptavidin.
  • Biotinylated antibodies targeting CD7 (T-lymphocytes), CD19 (B-lymphocytes), and CD16 (neutrophils/monocytes) were used for positive cell selection.
  • Leukocytes were incubated in coated tubes for 30 minutes at 23°C.

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Main Results:

  • Significant numbers of T-cells (31,500), B-cells (8,600), and CD16+ cells (23,600) were isolated from blood samples.
  • The presence of selected leukocytes had minimal impact on RT-PCR analysis of the bcr/abl translocation.
  • Leukocyte-free RT-PCR signal recovery was high (78-99%) even with residual cell populations.

Conclusions:

  • A reproducible method for obtaining eosinophil-free leukocyte populations for RT-PCR was developed.
  • This approach avoids the need for RNA extraction, simplifying the RT-PCR workflow.
  • The method offers a reasonable leukocyte yield and maintains RT-PCR assay integrity.