Detection of tumor messenger RNA in the serum of patients with malignant melanoma.

Author(s): Kopreski MS, Benko FA, Kwak LW, Gocke CD

Publication: Clin Cancer Res, 1999, Vol. 5, Page 1961-5

PubMed ID: 10473072 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of filtration, freeze-thaw cycling, and thaw duration on RT-PCR amplification of c-abl and tyrosinase in serum from patients with malignant melanoma and healthy controls. Serum from six patients with malignant melanoma was prepared within 4 h of blood draw and stored frozen at -70 degrees C for more than 2 years before RNA extraction. Serum was also obtained from 20 healthy patients within 4 h of blood draw, and RNA was extracted immediately. RNA was isolated with the Perfect RNA-Total RNA isolation kit. To verify that the presence of tyrosinase was not due to cellular contamination, 2 serum specimens were passed through a 0.45 um cellulose acetate filter. For freeze-thaw cycling experiments, serum was thawed on ice and refrozen at -20 degrees C.

   Summary of Findings:

   c-abl was detected in all specimens, while tyrosinase was detected in 4 of 6 specimens from patients with malignant melanoma and none of the 20 specimens from healthy patients. Passing the serum through a 0.45 um cellulose acetate filter to remove any intact cells had no effect on the detection of tyrosinase in serum, and the authors also report that filtration had no effect on c-abl detection. While tyrosinase levels were stable when RNA was extracted within 15 min of thawing, tyrosinase amplification was reduced after a second freeze-thaw cycle and abolished after a third freeze-thaw cycle or when specimens were thawed for 30 min or more. The authors report that adding RNAse inhibitor, but not guanidinium thiocyanate, to serum thawed twice allowed for detection of tyrosinase after 3 cycles but not 4. c-abl was reduced when serum from a healthy patient underwent a single freeze-thaw cycle compared to levels in fresh serum.

   Biospecimens

   • Bodily Fluid - Serum
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen
   • None (Fresh)

   Diagnoses:

   • Neoplastic - Melanoma
   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Filtration	0.45 um cellular acetate filter None
   Preaquisition	Diagnosis/ patient condition	Healthy Malignant melanoma
   Biospecimen Preservation	Type of fixation/preservation	Frozen None (fresh)
   Storage	Freeze/thaw cycling	0 cycles 1 cycle 2 cycles 3 cycles 4 cycles 5 cycles
   Storage	Thaw duration	0 min 15 min 30 min 45 min 60 min
   Analyte Extraction and Purification	RNase inactivation	Guanidinium thiocyanate RNAsin None
   RT-PCR Specific	Targeted nucleic acid	c-abl Tyrosinase

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