Simultaneous quantification of DNA copy number and transcripts by Selfie-dPCR

Petar Podlesniy 1,Ramon Trullas1
1Institute of Biomedical Research of Barcelona, Barcelona, Spain 

To understand the mechanisms that regulate DNA gene tran- scription and replication it is necessary to have a precise measurement of the number of transcripts per gene. However, in the case of mitochondrial DNA (mtDNA), precise measurement of mtDNA transcription requires to take into account that mtDNA is present in a number of copies that varies depending on cell type and conditions, it is expressed in a polycistronic transcript and both light and heavy chains express their own transcript in a dif- ferentially regulated way. The presence of two mtDNA encoded transcripts that are regulated independently prevents their use as reference transcripts. In addition, distinction between the expres- sion of the transcripts of the light and heavy chain is usually not achieved in the standard PCR workflow. Here we present a novel method named Selfie-PCR that allows the precise simulta- neous measurement of both genomic and mtDNA transcripts in the same sample. The number of transcripts per encoding gene can be assessed in a locus specific and strand specific manner. Selfie-PCR permits the quantification of transcription initiation events in both strands and the assessment of gene transcription progress. As this method uses genes present in the sample as the own reference stan- dards and does not rely on any external reference it can be used in cells and tissues from different origins with different gene copy number or metabolic state.
Acknowledgement: Supported by SAF2014-56644-R and CIBERNED PI2016/06-3 grants.

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Transferring qPCR methods into a droplet digital PCR format – Experiences from Official Food and Feed Control in GMO analysis

Sven Pecoraro 1,
1Bavarian Health and Food Safety Authority, Germany; 

According to Regulation (EC) No 1829/2003 genetically modi- fied organisms (GMO) have to be approved to be put on the market in the EU. Food or feed that contain, consist or are produced from GMO have to be properly labelled. This provision shall not apply up to a proportion of 0.9% of a given GMO material, provided that the presence of such material is adventitious or technically unavoid- able. From this it follows for official food and feed control, that if GMO material is qualitatively detected in a given sample the pro- portion of this material (taxon related) has to be quantified with qPCR. Quantification in GMO analysis consists of two independent qPCR reactions. In one reaction the amount of taxon (plant) spe- cific DNA is measured relative to a standard curve with known DNA copy numbers. The other reaction measures the DNA copy numbers of the specific GMO. These two DNA copy numbers are divided (cp gmo/cp reference gene) and multiplied by 100% to give the final GMO percentage (cp/cp). Typically the limit of quantifica- tion (LOQ) of qPCR in GMO analysis is 0.1%. According to Regulation (EU) No 619/2011 certain GMOs have to be accurately quantified at a GMO proportion of 0.1%. However, quantifying at the lower end of the dynamic range of qPCR can be inaccurate. Digital droplet PCR (ddPCR) offers the advantage that DNA copy numbers can be abso- lutely quantified without the need for standard curves. Additionally ddPCR is capable to accurately measure minor quantities of DNA. Furthermore ddPCR is considered as less susceptible to inhibitory effects which is relevant when analyzing DNA that derives from complex matrices like food or feed. Official control laboratories have to apply validated methods. In GMO analysis predominantly methods published by the Joint Research Centre (JRC) are used. When such qPCR methods are transferred into a ddPCR format several aspects should be considered. As usually the master mix is different in ddPCR than for qPCR, a selected set of GMO should be tested in order to assess the specificity of the reaction. In gen- eral the reaction conditions like annealing temperature, primer and probe concentrations should be taken as they were validated. If results are not satisfying, e.g. because the separation between posi- tive and negative signals is not optimal (intermediate signals = rain) then a temperature gradient can be performed to evaluate the most appropriate annealing temperature. One has to consider that if the annealing temperature is altered more than 1 ◦ C to 2 ◦ C (robustness range) then the method has possibly to be re-validated. Increased primer and/or probe concentrations can also lead to better signal separation. The experience of the GMO laboratory of the Bavarian Health and Food Safety Authority (LGL) with ddPCR is very promis- ing. The procedure is accurate even with minor quantities of DNA and serves as an alternative reference method to standard qPCR when quantifying GMO DNA derived from complex matrices.

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Monitoring EGFR mutations in lung cancer patients using 3-color Crystal Digital PCR

Rémi Dangla 1,
1Stilla Technologies, France; 

Presentation of the Naica System, a fast and user-friendly digital PCR system uniquely equipped with a 3-color readout capability for straightforward multiplexing. Application of the Naica System to monitor the treatment response of lung cancer patients from ctDNA, using a single 3-plex assay targeting both the sensitizing and resistance mutations. Preview on upcoming extended multiplexing capacity.

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Calibrating quantitative measurements of HIV DNA using digital PCR

Eloise Busby 1, Alexandra Whale1, R. Bridget Ferns 2, Paul R. Grant3, Gary Morley1, Jonathan Campbell1, Carole Foy1, Eleni Nastouli3,4, Jim Huggett1,5, Jeremy A. Garson 2,6
1 Molecular and Cell Biology Team, LGC, Teddington, UK
2 Department of Infection, Division of Infection and Immunity, University College London, London, UK
3 Department of Clinical Virology, University College London Hospital NHS Foundation Trust, and the UCL/UCLH NIHR Biomedical Research Centre, London, UK
4 Department of Population Policy and Practice, UCL GOS Institute of Child Health, London, UK
5 School of Biosciences & Medicine, Faculty of Health & Medical Science, University of Surrey, Guildford GU2 7XH, UK
6 National Transfusion Microbiology Laboratories, NHS Blood and Transplant, Colindale, London, UK 

Quantification of HIV DNA associated with the viral reservoir is increasingly used in research as a tool to study latent disease. Such a target could potentially be used clinically to assist moni- toring of disease progression or decline as it has been reported to correlate with viral outgrowth, and could serve as a biomarker for monitoring chronic infection. In addition to measurement of RNA viral load, qPCR is used as the method of choice for quantification of HIV DNA. Results are normalised to a human genomic target and reported as HIV DNA copies per 1,000,000 cells. When per- forming qPCR this is commonly achieved by calibrating against the 8E5 cell line reported to contain one stably integrated HIV genome per cell. In this study we used dPCR to investigate the stability of the HIV genome in the 8E5 cell line and demonstrated how accurate value assignment of this calibrator impacts upon qPCR measurement of HIV DNA. DNA from three separate sources of 8E5 cells, which included serially passaged fresh cells, was analysed using two droplet-based dPCR platforms. We identified genetic instability in the 8E5 cell line, which supports very recent find- ings of other researchers, and resulted in HIV DNA quantification in patient samples varying by a factor of ∼50. dPCR value assign- ment of the calibrators removed this error. Appropriately calibrated quantitative methods afford greater accuracy and measurement harmonisation when using qPCR to measure a specific sequence. We show here that dPCR is a method that can effectively be used to quantify calibration materials for this purpose.

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RT-Digital PCR for the evaluation of residual disease in chronic myeloid leukaemia

Mary Alikian 1,2,*,Olga Tatarinova1, Alistair Reid1, Jane Apperley2,3, Letizia Foroni2,3
1 Imperial Molecular Pathology, Imperial Healthcare Trust, Hammersmith Hospital, London, UK
2 Centre for Haematology, Faculty of Medicine, Imperial College London, London, UK
3 Clinical Haematology, Imperial College Healthcare NHS Trust, London, UK

Tyrosine Kinase Inhibitors (TKIs) are part of the successful clin- ical management of patients with Chronic Myeloid Leukaemia (CML). However, optimal clinical management of CML requires a robust, standardised laboratory assay used at key clinical mile- stones to ensure a successful outcome for patients on TKIs. Quantitative monitoring of %BCR-ABL1IS by reverse transcription quantitative PCR (RT-qPCR) is the gold standard strategy for evalu- ating patient response to therapy and classification into prognostic subgroups. However, it can be challenging to perform in a repro- ducible manner. Reverse-Transcription Digital PCR (RT-dPCR) is an adaptation of this method that could provide the robust and stan- dardised workflow needed for patient stratification.
Recent research from our lab showed that RT-dPCR, using the Europe Against Cancer (EAC) assay, did not significantly improve the sensitivity of residual disease quantification below Major Molecular Response (MMR). The presence of a considerable amount of background noise in the negative samples did not allow for an increase in the sensitivity. However, using RT-dPCR simplified the quantification process eliminating the need for using standard curves and enabling the absolute quantification of the BCR-ABL1 transcript molecules expressed as transcript copies per reaction.
In this study, we validated a new assay, called IDT, for the quan- tification of BCR-ABL1 transcripts on the RainDropTM dPCR platform. The IDT assay uses GUSB as a reference gene and is capable of quantifying the e13a2 and e14a2 transcript types of BCR-ABL1 inde- pendently in each sample in a multiplex format. Using this assay, we quantified MRD in 138 samples from 64 patients enrolled in the UK based De- Escalation and Stopping Treatment of Imatinib, Nilot- inib or sprYcel in Chronic Myeloid Leukaemia (DESTINY) trial and compared the BCR-ABL1 transcripts numbers quantified by both RT- qPCR and RT-dPCR. Although the IDT assay had significantly lower noise levels, the assay did not make a significant difference on the sensitivity compared to RT-qPCR. This result alludes to the fact that samples in deep molecular response are truly disease free.
In this work, we introduce the IDT assay coupled with RT-dPCR on the RainDrop platform for the quantification of BCR-ABL1 trans- cripts for monitoring MRD in CML patients.

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Liquid profiling of RNA and DNA for treatment response prediction and monitoring of cancer patients

Christof Winter 1,
1TU München, Germany; 

Cell-free nucleic acids released from dying tumor cells can readily be detected in the blood plasma. To distinguish tumor from normal cell-free nucleic acids, tumor-specific mutations can be used. Here I will present the results of two studies using this liq- uid profiling approach for tumor load monitoring and personalized treatment in cancer patients.
In breast cancer, where the tumor mutation spectrum differs substantially between patients, we make use of individual chro- mosomal rearrangements in the tumor that we detect and quantify in patient blood over time using personalized droplet digital PCR assays. This allowed to retrospectively detect a metastatic recur- rence in blood on average 11 months before clinical detection with symptoms. Early detection and early treatment might lead to improved patient survival.
In prostate cancer, the androgen receptor (AR) is an important target for hormone therapy (androgen deprivation therapy). How- ever, a considerable fraction of tumors expresses a splice variant (AR-V7) that is resistant to hormone therapy. We designed droplet digital PCR assays specific for AR and for AR-V7 and then quan- tified RNA levels of these transcripts in patient whole blood. In patients with metastatic prostate cancer, we found that high AR- V7 blood levels before therapy initiation were associated with poor progression-free survival and poor overall survival under hormone therapy. Notably, we found that none of the patients with high AR-V7 levels did respond to a subsequent hormone therapy. Trans- lating these results into routine clinical testing will contribute to precision oncology.

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Unprecedented accuracy when using digital PCR

Jim Huggett 1,
1LGC & University of Surrey, United Kingdom; 

Digital PCR (dPCR) offers a unique approach for molecular anal- ysis as it counts DNA molecules, separated by limiting dilution, in a defined volume. The method offers high quantitative precision and the fact that each DNA molecule has its own minute reaction (or partition) means rare genetic variants are easier to detect. The ability of dPCR to measure accurately (with both high precision and low bias) is of particular interest as it performs absolute quan- tification and thus may offer SI traceability. This could have wide ranging implication for other methods such as qPCR and NGS. This talk will present the research into dPCR accuracy and discuss some of the implications this may have on molecular measurement both in research and more routine applications.

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