Discussion board for any problem in quantitative real-time PCR.
BLOG of www.Gene-Quantification.info
- the reference in real time PCR, the academic and industrial information platform. The GQ pages describe and summarize all technical aspects involved in quantitative gene expression analysis using real-time qPCR & RT-qPCR.
qPCR, dPCR, NGS – A journey
Jim F. Huggett, Justin O’Grady, Stephen Bustin
Biomolecular Detection and Quantification, available
online 15 January 2015
Scientific conferences fulfill many roles, but one of the most
important ones is that they help shape the direction in which a
scientific discipline grows by promoting person-to-person exchanges of
information, ideas and constructive criticisms between scientists from
different backgrounds. This interaction also helps to identify areas of
controversy and promotes efforts to address and, it is hoped, resolve
them. This year is the 30th anniversary of the publication of the first
practical description of the polymerase chain reaction , arguably
one of the simplest and the most widely used molecular technology. It
also sees the 7th instalment of the
Freising PCR meetingshttp://www.qPCR-NGS-2015.net,
which are the longest established, continuous and most influential
conferences in this field and have provided a looking glass for
conceptual and technical innovation as well as practical applications
of PCR-associated methods.
We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates. In particular, how robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. We used six different qPCR instruments, we performed 1–16 qPCR replicates per concentration and we tested 2–10 μl volume of analyte transferred, respectively. We find that the estimated PCR efficiency varies significantly across different instruments. Using a Monte Carlo approach, we find the uncertainty in the PCR efficiency estimation may be as large as 42.5% (95% CI) if standard curve with only one qPCR replicate is used in 16 different plates. Based on our investigation we propose recommendations for the precise estimation of PCR efficiency: (1) one robust standard curve with at least 3–4 qPCR replicates at each concentration shall be generated, (2) the efficiency is instrument dependent, but reproducibly stable on one platform, and (3) using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range.
The small RNAs of bovine plasma and whole blood were analysed using next-generation sequencing to quantify, profile and compare the microRNAs (miRNA) and piRNA signatures in both bio fluids.
Evaluating read-count data resulted in a proportion of 5.0 ± 2.9 % of miRNAs in plasma while 38.2 ± 3.4 % were identified in whole blood. Regarding piRNAs, the percentages in both matrices were nearly the same: 1.4 ± 0.8 % of piRNAs in plasma and 1.9 ± 0.8 % in whole blood. Investigation of the ten most abundant miRNAs and piRNAs in both bio fluids revealed that two miRNAs and seven piRNAs were identical. Comparing the read-count values of these matching pairs highlighted that miRNA and piRNA levels in blood exceeded the abundance of their corresponding miRNAs and piRNAs in plasma, except liver-specific miR-122 and three piRNAs.
The data strengthened evidence that the circulating small RNA signature in plasma is not only influenced by hematocytes and certain small RNAs could originate from other sources than cellular blood components.
qPCR & NGS 2015 Symposium "Advanced Molecular Diagnostics for Biomarker Discovery" 7th international qPCR & NGS Event -- Symposium & Industrial Exhibition & Application Workshops 23-27 March 2015 TUM School of Life Sciences, Technical University of Munich, Germany Browse the Conference Agenda-- http://AgendaHTML.qPCR-NGS-2015.net Print agenda PDF versionhttp://AgendaPDF.qPCR-NGS-2015.net Keynote Lectures:
PCR in less than 30 seconds: Efficient, specific amplification with increased primer and polymerase. Carl T Wittwer; University of Utah, United States of America
Digital PLA (Proximity Ligation Assay) Stephen Bustin; Anglia Ruskin University, Chelmsford, Essex, United Kingdom
Next-Generation RNA-Seq. Gary Schroth; Illumina, United States of America
How could digital PCR benefit clinical analysis? Jim Francis Huggett; LGC, United Kingdom
Updated evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study. Jo Vandesompele; Ghent University on behalf of the microRNA quality control study consortium, Belgium
Mass Spectrometry Based Draft Of The Human Proteome. Bernhard Kuster; TU München, Freising Weihenstephan, Germany
Next Generation Sequencing Applications to the study of human and animal Microbiomes. Karen E. Nelson, J. Craig Venter Institute, United States of America
Expression Profiling of Circulating Tumor Cells: a Prognostic and Predictive Biomarker in Cancer. Mikael Kubista; TATAA Biocenter, Sweden
Variability of the Reverse Transcription Step: Practical Implications. Tania Nolan; The Gene Team, United Kingdom