Montag, 16. März 2015

qPCR, dPCR, NGS – A journey

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 [1], arguably one of the simplest and the most widely used molecular technology. It also sees the 7th instalment of the Freising PCR meetings http://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.

Donnerstag, 12. März 2015

How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments

How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments

David Svec, Ales Tichopad, Vendula Novosadova, Michael W. Pfaffl, Mikael Kubista
B
iomolecular Detection and Quantification (March 2015)

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.

Dienstag, 24. Februar 2015

Comparison of the miRNome and piRNome of bovine blood and plasma by small RNA sequencing.

Comparison of the miRNome and piRNome of bovine blood and plasma by small RNA sequencing.

OBJECTIVES:

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.

RESULTS:

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.

CONCLUSIONS:

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.

Samstag, 14. Februar 2015

Keynote Lectures -- Browse the qPCR & NGS 2015 Conference Agenda


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 version  http://AgendaPDF.qPCR-NGS-2015.net

Keynote Lectures:
  1. PCR in less than 30 seconds: Efficient, specific amplification with increased primer and polymerase.
    Carl T Wittwer; University of Utah, United States of America
  2. Digital PLA  (Proximity Ligation Assay)
    Stephen Bustin; Anglia Ruskin University, Chelmsford, Essex, United Kingdom
  3. Next-Generation RNA-Seq.
    Gary Schroth; Illumina, United States of America
  4. How could digital PCR benefit clinical analysis?
    Jim Francis Huggett; LGC, United Kingdom
  5. 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
  6. Mass Spectrometry Based Draft Of The Human Proteome.
    Bernhard Kuster; TU München, Freising Weihenstephan, Germany 
  7. Next Generation Sequencing Applications to the study of human and animal Microbiomes.
    Karen E. Nelson, J. Craig Venter Institute, United States of America 
  8. Expression Profiling of Circulating Tumor Cells: a Prognostic and Predictive Biomarker in Cancer.
    Mikael Kubista; TATAA Biocenter, Sweden
  9. Variability of the Reverse Transcription Step: Practical Implications.
    Tania Nolan; The Gene Team, United Kingdom
  10. Decoding lncRNA functions using high-throughput pathway perturbation.
    Pieter Mestdagh; Biogazelle, Belgium

-----------------------------------

Event Promotion Trailer  http://youtu.be/44nJ43gzXJQ
Download the event flyer  http://event-announcement.qPCR-NGS-2015.net
Event location on Google maps  http://goo.gl/maps/3p3gx
Register for Symposium & Workshops  http://registration.qPCR-NGS-2015.net

Mittwoch, 28. Januar 2015

Class I odorant receptors, TAS1R and TAS2R taste receptors, are markers for subpopulations of circulating leukocytes.

Class I odorant receptors, TAS1R and TAS2R taste receptors, are markers for subpopulations of circulating leukocytes.
 2015 Jan 26. pii: jlb.2A0714-331RR. [Epub ahead of print]
Our cellular immune system has to cope constantly with foodborne substances that enter the bloodstream postprandially. Here, they may activate leukocytes via specific but yet mostly unknown receptors. Ectopic RNA expression out of gene families of chemosensory receptors, i.e., the ∼400 ORs, ∼25 TAS2R bitter-taste receptors, and the TAS1R umami- and sweet-taste receptor dimers by which we typically detect foodborne substances, has been reported in a variety of peripheral tissues unrelated to olfaction or taste. In the present study, we have now discovered, by gene-specific RT-PCR experiments, the mRNA expression of most of the Class I ORs (TAS1R) and TAS2R in 5 different types of blood leukocytes. Surprisingly, we did not detect Class II OR mRNA. By RT-qPCR, we show the mRNA expression of human chemosensory receptors and their cow orthologs in PMN, thus suggesting an evolutionary concept. By immunocytochemistry, we demonstrate that some olfactory and taste receptors are expressed, on average, in 40-60% of PMN and T or B cells and largely coexpress in the same subpopulation of PMN. The mRNA expression and the size of subpopulations expressing certain chemosensory receptors varied largely among individual blood samples, suggesting a regulated expression of olfactory and taste receptors in these cells. Moreover, we show mRNA expression of their downstream signaling molecules and demonstrate that PTX abolishes saccharin- or 2-PEA-induced PMN chemotactic migration, indicating a role for Gi-type proteins. In summary, our data suggest "chemosensory"-type subpopulations of circulating leukocytes.