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Detection of microRNAs in archival cytology urine smears.

Simonato F, Ventura L, Sartori N, Cappellesso R, Fassan M, Busund LT, Fassina A - PLoS ONE (2013)

Bottom Line: Only samples with >5,000 urothelial cells and with <50% of inflammatory cells/red blood cells clusters were considered.Overall, a satisfactory amount of total RNA was obtained from all the considered samples (mean 1.27±1.43 µg, range 0.06-4.60 µg).Further studies should investigate if miRNAs profiling can be successfully translated into clinical practice as diagnostic or prognostic markers.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine (DIMED), Surgical Pathology & Cytopathology Unit, University of Padua, Padua, Italy.

ABSTRACT
MicroRNAs' dysregulation and profiling have been demonstrated to be clinically relevant in urothelial carcinoma (UC). Urine cytology is commonly used as the mainstay non-invasive test for secondary prevention and follow-up of UC patients. Ancillary tools are needed to support cytopathologists in the diagnosis of low-grade UC. The feasibility and reliability of microRNAs profiling by qRT-PCR analysis (miR-145 and miR-205) in archival routine urine cytology smears (affected by fixation/staining [Papanicolau] and room temperature storage) was tested in a series of 15 non-neoplastic and 10 UC urine specimens. Only samples with >5,000 urothelial cells and with <50% of inflammatory cells/red blood cells clusters were considered. Overall, a satisfactory amount of total RNA was obtained from all the considered samples (mean 1.27±1.43 µg, range 0.06-4.60 µg). Twenty nanograms of total RNA have been calculated to be the minimal total RNA concentration for reliable and reproducible miRNAs expression profiling analysis of archival cytological smears (slope= -3.4084; R-squared=0.99; efficiency=1.94). miR-145 and miR-205 were significantly downregulated in UC samples in comparison to non-tumor controls. These findings demonstrate that urine archival cytology smears are suitable for obtaining high-quality RNA to be used in microRNAs expression profiling. Further studies should investigate if miRNAs profiling can be successfully translated into clinical practice as diagnostic or prognostic markers.

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Related in: MedlinePlus

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.
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pone-0057490-g002: 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.

Mentions: To estimate the efficiency e of the qRT-PCR obtained by cytology smear and to test the relationship between Ct and ldose, a covariance analysis (ANCOVA) model was applied to the two cell lines (Capan-1 and BxPC3). The best cut-off for RNA amount to obtain a proper qRT-PCR analysis was selected in terms of the estimated slope (Table 2). All the diagnostics supported the estimated ANCOVA models. For RNA U6B, when considering all the tested dilutions (i.e., 50 ng, 40 ng, 30 ng, 20 ng, 10 ng, 5 ng, 1 ng, 0.1 ng), the ANCOVA analysis (R2 = 0.99) revealed that the slope of the relationship between ldose and Ct was the same in the two cell lines (p = 0.55) and it was equal to −3.73 (±0.07, p<0.0001), with efficiency e = 1.70. The Ct measurements were systematically lower in Capan-1 compared with BxPC3 (-2.68±0.12, p<0.0001). When considering amount of total RNA ≥1 ng, the ANCOVA analysis (R2 = 0.99) showed that the slope of the relationship between ldose and Ct was equal to −3.561 (±0.11, p<.0001), with efficiency e = 1.81. The estimated models are Ct = 27.65–3.55*ldose for BxPc3 and Ct = 24.97–3.55*ldose for Capan-1 (Figure 2A). For miR-205, when considering all the dilutions, ANCOVA analysis (R2 = 0.98) revealed that the slope of the relationship between ldose and Ct was the same in the two cell lines and was equal to −4.00 (±0.12, p<.0001), with efficiency e = 1.50. When considering amount of total RNA ≥5 ng, the ANCOVA model (R2 = 0.91) showed that the slope of the relationship between ldose and Ct was −3.649 (±0.39, p<.000), with efficiency e = 1.75. The estimated model was Ct = 29.09–3.64*ldose both for BxPC3 and Capan-1 (Figure 2B).


Detection of microRNAs in archival cytology urine smears.

Simonato F, Ventura L, Sartori N, Cappellesso R, Fassan M, Busund LT, Fassina A - PLoS ONE (2013)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585351&req=5

pone-0057490-g002: 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.
Mentions: To estimate the efficiency e of the qRT-PCR obtained by cytology smear and to test the relationship between Ct and ldose, a covariance analysis (ANCOVA) model was applied to the two cell lines (Capan-1 and BxPC3). The best cut-off for RNA amount to obtain a proper qRT-PCR analysis was selected in terms of the estimated slope (Table 2). All the diagnostics supported the estimated ANCOVA models. For RNA U6B, when considering all the tested dilutions (i.e., 50 ng, 40 ng, 30 ng, 20 ng, 10 ng, 5 ng, 1 ng, 0.1 ng), the ANCOVA analysis (R2 = 0.99) revealed that the slope of the relationship between ldose and Ct was the same in the two cell lines (p = 0.55) and it was equal to −3.73 (±0.07, p<0.0001), with efficiency e = 1.70. The Ct measurements were systematically lower in Capan-1 compared with BxPC3 (-2.68±0.12, p<0.0001). When considering amount of total RNA ≥1 ng, the ANCOVA analysis (R2 = 0.99) showed that the slope of the relationship between ldose and Ct was equal to −3.561 (±0.11, p<.0001), with efficiency e = 1.81. The estimated models are Ct = 27.65–3.55*ldose for BxPc3 and Ct = 24.97–3.55*ldose for Capan-1 (Figure 2A). For miR-205, when considering all the dilutions, ANCOVA analysis (R2 = 0.98) revealed that the slope of the relationship between ldose and Ct was the same in the two cell lines and was equal to −4.00 (±0.12, p<.0001), with efficiency e = 1.50. When considering amount of total RNA ≥5 ng, the ANCOVA model (R2 = 0.91) showed that the slope of the relationship between ldose and Ct was −3.649 (±0.39, p<.000), with efficiency e = 1.75. The estimated model was Ct = 29.09–3.64*ldose both for BxPC3 and Capan-1 (Figure 2B).

Bottom Line: Only samples with >5,000 urothelial cells and with <50% of inflammatory cells/red blood cells clusters were considered.Overall, a satisfactory amount of total RNA was obtained from all the considered samples (mean 1.27±1.43 µg, range 0.06-4.60 µg).Further studies should investigate if miRNAs profiling can be successfully translated into clinical practice as diagnostic or prognostic markers.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine (DIMED), Surgical Pathology & Cytopathology Unit, University of Padua, Padua, Italy.

ABSTRACT
MicroRNAs' dysregulation and profiling have been demonstrated to be clinically relevant in urothelial carcinoma (UC). Urine cytology is commonly used as the mainstay non-invasive test for secondary prevention and follow-up of UC patients. Ancillary tools are needed to support cytopathologists in the diagnosis of low-grade UC. The feasibility and reliability of microRNAs profiling by qRT-PCR analysis (miR-145 and miR-205) in archival routine urine cytology smears (affected by fixation/staining [Papanicolau] and room temperature storage) was tested in a series of 15 non-neoplastic and 10 UC urine specimens. Only samples with >5,000 urothelial cells and with <50% of inflammatory cells/red blood cells clusters were considered. Overall, a satisfactory amount of total RNA was obtained from all the considered samples (mean 1.27±1.43 µg, range 0.06-4.60 µg). Twenty nanograms of total RNA have been calculated to be the minimal total RNA concentration for reliable and reproducible miRNAs expression profiling analysis of archival cytological smears (slope= -3.4084; R-squared=0.99; efficiency=1.94). miR-145 and miR-205 were significantly downregulated in UC samples in comparison to non-tumor controls. These findings demonstrate that urine archival cytology smears are suitable for obtaining high-quality RNA to be used in microRNAs expression profiling. Further studies should investigate if miRNAs profiling can be successfully translated into clinical practice as diagnostic or prognostic markers.

Show MeSH
Related in: MedlinePlus