Figure 1.
Selection of the cytology specimens.
Only cases with >5,000 well-preserved and well-visualized urothelial cells (as observed in 10 fields at 4x magnification) were selected. Cases largely constituted by red blood cells or inflammatory cells were excluded.
Table 1.
Cytological and molecular features of the 25 cytology samples.
Figure 2.
Identification of the minimal RNA quantity to obtain an adequate qRT-PCR reaction from cytology specimens.
(A) The relationship between Ct and ldose for the two cell lines (BxPc3 and Capan-1) was analyzed as covariance (ANCOVA) model and the minimal RNA quantity was identified in terms of the estimated slope. For RNU6B, the ANCOVA analysis (R2 = 0.99) revealed that the slope of the relationship between ldose and Ct is the same in the two cells lines (p = 0.55) and it is equal to −3.73 (±0.07, p<0.0001), with efficiency e = 1.70. The estimated models are Ct = 27.65–3.55*ldose for BxPc3 and Ct = 24.97–3.55*ldose for Capan-1. (B) For miR-205 ANCOVA analysis (R2 = 0.98) revealed that the slope of the relationship between ldose and Ct is the same in the two cell lines and is equal to −4.00 (±0.12, p<0.000), with efficiency e = 1.50. The estimated model is Ct = 29.09–3.64*ldose both for BxPc3 and Capan-1. (C) To estimate the relationship between Ct and ldose on the 15 non-neoplastic patients, we used linear mixed effect models including a random effect for the subject. (D) The minimal RNA quantity was selected in terms of the fixed effect slope. For miR-205, when considering all the dilutions, the LME analysis (R2 = 0.92) revealed that the fixed effect slope of the relationship between ldose and Ct is −1.73 (±0.13, p<0.000), with efficiency e = 4.78.
Table 2.
RNA amount cut-off values for qRT-PCR analysis using archival cytology smears.
Figure 3.
Total RNA extracted from archival urine cytology smears is suitable for miRNA expression profiling.
Box plots show differences in miRNAs expression between non-tumor (NT) and low grade urothelial carcinoma samples (T). *p<0.05.