The Effect of Centrifugal Force in Quantification of Colorectal Cancer-Related mRNA in Plasma Using Targeted Sequencing

In our previous study, we detected the effects of centrifugal forces on plasma RNA quantification by quantitative reverse transcription PCR. The aims of this study were to perform targeted mRNA sequencing and data analysis in healthy donors' plasma prepared by two centrifugation protocols and to investigate the effects of centrifugal forces on plasma mRNA quality and quantity. Targeted mRNA sequencing was performed using a custom panel with 108 colorectal cancer-related genes in 18 healthy donors' plasma that prepared by (1) 3,500 g for 10 min at 4°C and (2) 1,600 g for 10 min at 4°C followed by 16,000 g for 10 min at 4°C. Results showed that plasma ribosomal RNA was detected in 16/18 (88.9%) 3,500 g and 6/18 (33.3%) 1,600 g followed by 16,000 g centrifuged plasma. For targeted sequencing, 75/108 (69.4%) and 86/108 (79.6%) genes were detected in 3,500 and 1,600 g followed by 16,000 g, respectively, while 16/108 (14.8%) genes were not detected in both centrifugations. Detailed analysis showed that 2 of 108 (1.85%) genes showed lower expressions in 3,500 g than in 1,600 g followed by 16,000 g. The median expressions of genes in 3,500 g were positively correlated with the expressions in 1,600 g followed by 16,000 g (R2 = 0.9471, P < 0.0001, Spearman rank correlation). Meanwhile, plasma samples were not distinctively clustered based on centrifugal forces according to hierarchical clustering. Targeted mRNA sequencing and subsequent data analysis were performed in this study to investigate the effects of two different centrifugal forces that are commonly used in plasma collection. Our targeted sequencing results help to understand the centrifugal force effects on plasma mRNA, and these findings show that the centrifugation protocol for plasma mRNA research using targeted sequencing can be standardized which facilitates multicenter studies for comparison and quality assurance in the future

Image_1_The Effect of Centrifugal Force in Quantification of Colorectal Cancer-Related mRNA in Plasma Using Targeted Sequencing.PDF

<p>In our previous study, we detected the effects of centrifugal forces on plasma RNA quantification by quantitative reverse transcription PCR. The aims of this study were to perform targeted mRNA sequencing and data analysis in healthy donors' plasma prepared by two centrifugation protocols and to investigate the effects of centrifugal forces on plasma mRNA quality and quantity. Targeted mRNA sequencing was performed using a custom panel with 108 colorectal cancer-related genes in 18 healthy donors' plasma that prepared by (1) 3,500 g for 10 min at 4°C and (2) 1,600 g for 10 min at 4°C followed by 16,000 g for 10 min at 4°C. Results showed that plasma ribosomal RNA was detected in 16/18 (88.9%) 3,500 g and 6/18 (33.3%) 1,600 g followed by 16,000 g centrifuged plasma. For targeted sequencing, 75/108 (69.4%) and 86/108 (79.6%) genes were detected in 3,500 and 1,600 g followed by 16,000 g, respectively, while 16/108 (14.8%) genes were not detected in both centrifugations. Detailed analysis showed that 2 of 108 (1.85%) genes showed lower expressions in 3,500 g than in 1,600 g followed by 16,000 g. The median expressions of genes in 3,500 g were positively correlated with the expressions in 1,600 g followed by 16,000 g (R² = 0.9471, P < 0.0001, Spearman rank correlation). Meanwhile, plasma samples were not distinctively clustered based on centrifugal forces according to hierarchical clustering. Targeted mRNA sequencing and subsequent data analysis were performed in this study to investigate the effects of two different centrifugal forces that are commonly used in plasma collection. Our targeted sequencing results help to understand the centrifugal force effects on plasma mRNA, and these findings show that the centrifugation protocol for plasma mRNA research using targeted sequencing can be standardized which facilitates multicenter studies for comparison and quality assurance in the future.</p

Table_1_The Effect of Centrifugal Force in Quantification of Colorectal Cancer-Related mRNA in Plasma Using Targeted Sequencing.PDF

<p>In our previous study, we detected the effects of centrifugal forces on plasma RNA quantification by quantitative reverse transcription PCR. The aims of this study were to perform targeted mRNA sequencing and data analysis in healthy donors' plasma prepared by two centrifugation protocols and to investigate the effects of centrifugal forces on plasma mRNA quality and quantity. Targeted mRNA sequencing was performed using a custom panel with 108 colorectal cancer-related genes in 18 healthy donors' plasma that prepared by (1) 3,500 g for 10 min at 4°C and (2) 1,600 g for 10 min at 4°C followed by 16,000 g for 10 min at 4°C. Results showed that plasma ribosomal RNA was detected in 16/18 (88.9%) 3,500 g and 6/18 (33.3%) 1,600 g followed by 16,000 g centrifuged plasma. For targeted sequencing, 75/108 (69.4%) and 86/108 (79.6%) genes were detected in 3,500 and 1,600 g followed by 16,000 g, respectively, while 16/108 (14.8%) genes were not detected in both centrifugations. Detailed analysis showed that 2 of 108 (1.85%) genes showed lower expressions in 3,500 g than in 1,600 g followed by 16,000 g. The median expressions of genes in 3,500 g were positively correlated with the expressions in 1,600 g followed by 16,000 g (R² = 0.9471, P < 0.0001, Spearman rank correlation). Meanwhile, plasma samples were not distinctively clustered based on centrifugal forces according to hierarchical clustering. Targeted mRNA sequencing and subsequent data analysis were performed in this study to investigate the effects of two different centrifugal forces that are commonly used in plasma collection. Our targeted sequencing results help to understand the centrifugal force effects on plasma mRNA, and these findings show that the centrifugation protocol for plasma mRNA research using targeted sequencing can be standardized which facilitates multicenter studies for comparison and quality assurance in the future.</p

Human Papillomavirus DNA Detection in Menstrual Blood from Patients with Cervical Intraepithelial Neoplasia and Condyloma Acuminatum ▿

The Papanicolaou test generates pain and embarrassment, and cytology screening has limited sensitivity for detection of cervical neoplasia. These factors urge the use of another screening test that can overcome these limitations. We explore a completely noninvasive method using detection of human papillomavirus (HPV) DNA in women's menstrual blood (MB). The participants were divided into 3 cohorts: (i) 235 patients with cervical intraepithelial neoplasia 3 (CIN 3) (n = 48), CIN 2 (n = 60), CIN 1 (n = 58), or condyloma acuminatum (CAC) (n = 69) before treatment or remission; (ii) from the first cohort of patients, 108 CIN 3 or CIN 2 patients after treatment and 62 CIN 1 or CAC patients after remission; and (iii) 323 apparently normal subjects (ANS) without any cervical disease. The HPV genotypes of the infected patients were confirmed by direct sequencing. Quantitative real-time PCR (QRT-PCR) was used to measure the MB HPV16 load for 15 infected patients. Results showed that the sensitivity, specificity, and positive and negative predictive values for detection of MB HPV DNA in samples from patients with CIN or CAC were 82.8%, 93.1%, 90.0%, and 87.9%, respectively. Moreover, MB HPV DNA was found in samples from 22.2% of CIN 3 or CIN 2 patients after treatment, 0.0% of CIN 1 or CAC patients after remission, and 8.1% of ANS, 4 of whom were found to have CIN 1 or CAC. Furthermore, QRT-PCR showed that the normalized MB HPV16 DNA copy numbers in samples from patients with CIN 1 to CIN 3 were significantly increased. These preliminary results suggested that MB HPV DNA is a potential noninvasive marker for these premalignant cervical diseases

FZD3 expression in various non-CRC metastatic carcinomas.

<p>(A) Percentage of various non-CRC metastatic carcinomas with FZD3 ICC staining and (B) ICC scores of FZD3 in various non-CRC metastatic carcinomas. (HCC = hepatocellular carcinoma, RCCC = renal clear cell carcinoma, SCC1 = squamous cell carcinoma, LAC = lung adenocarcinoma, LNSCC = lung non-small cell carcinoma, PTC = papillary thyroid carcinoma, BC = breast carcinoma, OCCC = ovary clear cell carcinoma, CNSCC = cervical non-small cell carcinoma, FTSAC = fallopian tube serous adenocarcinoma, TCC = transitional cell carcinoma, SCC2 = small cell carcinoma and CND = carcinoma with neuroendocrine differentiation).</p

ICC scores of FZD3 in various Dukes stage of patients with CRC.

<p>ICC scores of FZD3 in various Dukes stage of patients with CRC.</p

ICC staining of FZD3 in (A) a lymph node with CRC metastasis from a Dukes 3 patient and (B) an ovary with CRC metastasis from a Dukes 4 patient (T = tumor, Lym = lymphoid cells, original magnification, X400, scale bar = 100 µm).

<p>ICC staining of FZD3 in (A) a lymph node with CRC metastasis from a Dukes 3 patient and (B) an ovary with CRC metastasis from a Dukes 4 patient (T = tumor, Lym = lymphoid cells, original magnification, X400, scale bar = 100 µm).</p

ICC scores of FZD3 in (A) adenomas with synchronous carcinomas (AWSC) and (B) pure adenomas.

<p>ICC scores of FZD3 in (A) adenomas with synchronous carcinomas (AWSC) and (B) pure adenomas.</p

ICC staining of FZD3 in (A) CRC, (B), CAD, (C) colorectal polyp, (D) normal colorectal tissues, (E) positive control and (F) negative control (Original magnification, X400, scale bar = 100 µm).

<p>ICC staining of FZD3 in (A) CRC, (B), CAD, (C) colorectal polyp, (D) normal colorectal tissues, (E) positive control and (F) negative control (Original magnification, X400, scale bar = 100 µm).</p

Overall survival analyses for the 1^st cohort of 186 Dukes A to D CRC patients (A) stratified by a median FZD3 ICC score of 327, (B) with high and low FZD3 expression and (C) with moderately high and moderately low FZD3 expression.

<p>Overall survival analyses for the 1^st cohort of 186 Dukes A to D CRC patients (A) stratified by a median FZD3 ICC score of 327, (B) with high and low FZD3 expression and (C) with moderately high and moderately low FZD3 expression.</p