Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Direct serum assay for microRNA-21 concentrations in early and advanced breast cancer.

Author(s): Asaga S, Kuo C, Nguyen T, Terpenning M, Giuliano AE, Hoon DS

Publication: Clin Chem, 2011, Vol. 57, Page 84-91

PubMed ID: 21036945 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of the study was to optimize a direct amplification protocol for quantification of miR-21 in serum, investigate the stability of miR-21 during freeze-thaw cycling of serum, and to investigate the sensitivity and specificity of this assay for the diagnosis of breast cancer. Levels of miR-21 in FFPE tumor and normal adjacent specimens were also compared. Blood was collected from 102 patients with breast cancer and 20 healthy women in red tiger-top gel separator tubes. After clotting for 2-5 h, serum was obtained by centrifugation (details not included) and use of a 13-mm serum filter and then stored frozen at -80°C. RNA was extracted from serum using TRI reagent BD and reverse transcribed using miR-21 specific primers. In 12 cases, RNA extraction was not performed and instead serum was mixed with: 1) nothing, 2) Proteinase K, 3) 1% Tween-20 and Proteinase K, 4) 2.5% Tween-20 and Proteinase K, 5) 1% Tween-20, or 6) 2.5% Tween-20 before reverse transcription and then centrifugation at 9000 x g for 5 min before real-time PCR amplification. The effect of DNAse treatment was tested on eight serum specimens. RNA was extracted from fresh 10 µm sections of 14 matched archival FFPE breast tumor and normal adjacent specimens using a modification of the RNAWiz Isolation Kit and quantified using Quant-iT RiboGreen RNA Assay Kit. miR-21was quantified by real-time RT-PCR and was normalized to RNU6B (tissues) or miR-16 (serum). The stability study was conducted using four serum specimens that were subjected to up to four cycles of freezing at -80°C and thawing at 23°C.

   Summary of Findings:

   miR-21 was only amplified directly in serum when the serum was treated with Tween-20 in the absence of Proteinase K. miR-21 levels -21 levels were strongly correlated specimens in which RNA was extracted before analysis and when directly amplified from serum after treatment with 2.5% Tween-20 (r=0.796) but no correlation was observed when amplified directly from serum treated with 1% Tween-20(r=-0.064). Using direct amplification of serum treated with 2.5% Tween-20, there was no difference in miR-16 normalized miR-21 levels when 1.25 µL or 0.625 µL of serum were used but miR-16 was not detected when 0.3125 µL of serum was used. The mean standard deviation (SD) of the quantification cycle (Cq) values in this assay ranged from 0.42-0.59. The authors report that DNAse treatment had little effect on the Cq values (mean of 0.75 cycles). The normalized levels of miR-21 were not significantly affected by up to four freeze-thaw cycles. No difference in miR-16 Cq values were found among serum from healthy controls and different stages of breast cancer regardless of if RNA was extracted prior to analysis or amplified directly from serum. In contrast, normalized miR-21 levels were significantly higher in serum from breast cancer patients than controls, regardless of stage and RNA extraction (P<0.001). While no differences in serum miR-21 levels were found among the different cancer stages when RNA was extracted from serum, significantly higher normalized miR-21 levels were found in the serum of stage IV cancer patients than stage I (P=0.035 and P<0.001), II (P=0.0034 and P<0.001), and III (P=0.0014 and P=0.0006) patients when amplified directly from serum in two different cohorts. Importantly, the miR-21 direct amplification assay had a specificity of 75% and a sensitivity of 67% for diagnosis of locoregional breast cancer and a specificity of 86% and sensitivity of 59% for diagnosis of stage IV breast cancer versus stage I, II, or III. Levels of miR-21 were found to be higher in FFPE breast tumor than in normal adjacent tissue.

   Biospecimens

   • Bodily Fluid - Serum
   • Tissue - Breast

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Normal
   • Neoplastic - Normal Adjacent

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Breast Cancer Healthy
   Preaquisition	Prognostic factor	Stage I breast cancer Stage II breast cancer Stage III breast cancer Stage IV breast cancer
   Biospecimen Aliquots and Components	Aliquot size/volume	0.3125 µL 0.625 µL 1.25 µL
   Analyte Extraction and Purification	Analyte isolation method	TRI reagent BD Addition of Tween-20
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-21 miR-16 RNU6B
   Analyte Extraction and Purification	Protein digestion	Proteinase K
   Analyte Extraction and Purification	Protein solubilization	None 1% Tween-20 2.5% Tween-20
   Storage	Freeze/thaw cycling	1 cycle 2 cycles 3 cycles 4 cycles
   Analyte Extraction and Purification	Nucleic acid digestion	DNAse treated Not treated

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