On non-detects in qPCR data

McCall, Matthew N.; McMurray, Helene R.; Land, Hartmut; Almudevar, Anthony

doi:10.1093/bioinformatics/btu239

Cited by 152 publications

(180 citation statements)

References 20 publications

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“…The rationale for this threshold for expression positivity is provided in the Supplementary material. Undetermined values of expressions (Ct ≥ 40) for this set of miRNAs were imputed by EM-based model of the missing data mechanism [24]. The individual Ct values for target miRNAs were normalised against the geometrical mean of the Ct values of U6 RNA, RNU44 and RNU48, and nine most stably expressed miRNAs that were determined with use of the NormFinder application (Appendix A, Table 1 in Supplementary material) [25].…”

Section: Methodsmentioning

confidence: 99%

Prognostic value of 5-microRNA based signature in T2-T3N0 colon cancer

Bobowicz

Skrzypski

Czapiewski

et al. 2016

Clin Exp Metastasis

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The role of adjuvant chemotherapy in stage T2-T3N0 colon cancer (CC) is controversial and there are currently no reliable factors allowing for individual selection of patients with high risk of relapse for such therapy. We searched for microRNA-based signature with prognostic significance in this group. We assessed by qRT-PCR expression of 754 microRNAs (miRNAs) in tumour samples from 85 stage pT2-3N0 CC patients treated with surgery alone. MiRNA expression was compared between two groups of patients: 40 and 45 patients who did and did not develop distant metastases after resection, respectively. Additionally, miRNA expression was compared between CC and normal colon mucosa samples and between the mismatch repair (MMR) competent and deficient tumours. Low expression of miR-1300 and miR-939 was significantly correlated with shorter distant metastasis-free survival (DMFS) in Cox univariate analysis (p.adjusted = 0.049). The expression signature of five miRNAs (miR-1296, miR-135b, miR-539, miR-572 and miR-185) was found to be prognostic [p = 1.28E−07, HR 8.4 (95 % CI: 3.81–18.52)] for DMFS and cross-validated in a leave-one-out analysis, with the sensitivity and specificity of 74 and 78 %, respectively. The expression of miR-592 was significantly associated with the MMR status (p.adjusted <0.01). The expression of several novel miRNAs were found to be tumour specific, e.g. miR-888, miR-523, miR-18b, miR-302a, miR-423-5p, miR-582-3p (p < 0.05). We developed a miRNA expression signature that may be predictive for the risk of distant relapse in early stage CC and confirmed previously reported association between miR-592 expression and MMR status.Electronic supplementary materialThe online version of this article (doi:10.1007/s10585-016-9810-1) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Prognostic value of 5-microRNA based signature in T2-T3N0 colon cancer

Bobowicz

Skrzypski

Czapiewski

et al. 2016

Clin Exp Metastasis

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“…To avoid bias introduced by the issue of non-detects [14], we employed a left-censoring statistical approach to determine per-target differential expression in cases versus controls. Table 2 provides univariate differential expression results for each biomarker, ranked by Tobit model [15] P -value.…”

Section: Resultsmentioning

confidence: 99%

Urinary mRNA biomarker panel for the detection of urothelial carcinoma

Urquidi¹,

Netherton²,

Gomes-Giacoia³

et al. 2016

Oncotarget

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The early detection of bladder cancer is important as the disease has a high rate of recurrence and progression. The development of accurate, non-invasive urinary assays would greatly facilitate detection. In previous studies, we have reported the discovery and initial validation of mRNA biomarkers that may be applicable in this context. In this study, we evaluated the diagnostic performance of proposed molecular signatures in an independent cohort.Forty-four mRNA transcripts were monitored blindly in urine samples obtained from a cohort of 196 subjects with known bladder disease status (89 with active BCa) using quantitative real-time PCR (RT-PCR). Statistical analyses defined associations of individual biomarkers with clinical data and the performance of predictive multivariate models was assessed using ROC curves. The majority of the candidate mRNA targets were confirmed as being associated with the presence of BCa over other clinical variables. Multivariate models identified an optimal 18-gene diagnostic signature that predicted the presence of BCa with a sensitivity of 85% and a specificity of 88% (AUC 0.935). Analysis of mRNA signatures in naturally micturated urine samples can provide valuable information for the evaluation of patients under investigation for BCa. Additional refinement and validation of promising multi-target signatures will support the development of accurate assays for the non-invasive detection and monitoring of BCa.

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“…Thermal cycling conditions were as follows: 95℃ for 10 min, 40 cycles of 95℃ for 15 s and 60℃ for 30 s. Reactions were conducted in triplicate, including a no template control and positive control. To account for nondetects in the qPCR data, C t values >35 were replaced with a C t of 35, which has been previously shown to reduce bias [14]. Ion Torrent NGS Protocol Library generation followed the protocol described in the Ion AmpliSeq Library Kit 2.0 User Guide using the Ion AmpliSeq Cancer Hotspot Panel v2 primer pool, comprising 207 amplicons covering approximately 2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes.…”

Section: Pcr Primers and Probesmentioning

confidence: 99%

Peptide nucleic acid clamping to improve the sensitivity of Ion Torrent-based detection of an oncogenic mutation in <em>KRAS</em>

Rakhit

Ottolini

Jones

et al. 2017

Matters

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Detecting oncogenic changes in the genome of cancer patients is crucial for targeted therapy. Such changes include alterations to KRAS, a GTPase located upstream of several signalling transduction pathways implicated in cancer formation. While nextgeneration sequencing (NGS) allows for comprehensive analysis of a genome, the technology can struggle to detect low frequency variants. To improve the sensitivity of NGS for detecting mutations we created a peptide nucleic acid (PNA) clamp, validated by qPCR, designed to bind wild-type KRAS (WT KRAS) across codon 12 during the PCR amplification stage of a NGS library preparation. We tested the effect of clamping the wild-type KRAS sequence in a reference standard with a KRAS c.35G>A mutation (KRAS G12D ) at an allelic frequency (AF) of 1.3% and on circulating-free DNA from a patient harbouring a KRAS G12D mutation (at an AF of 3.2%). Runs were conducted using 10, 5, 2.5 and 1 ng of DNA input. The PNA increased the number of mutant reads and their frequency relative to wild-type calls, allowing for more sensitive detection at all tested concentrations of DNA input.

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On non-detects in qPCR data

Cited by 152 publications

References 20 publications

Prognostic value of 5-microRNA based signature in T2-T3N0 colon cancer

Prognostic value of 5-microRNA based signature in T2-T3N0 colon cancer

Urinary mRNA biomarker panel for the detection of urothelial carcinoma

Peptide nucleic acid clamping to improve the sensitivity of Ion Torrent-based detection of an oncogenic mutation in <em>KRAS</em>

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