RT-droplet Digital PCR Research Articles

DMF-Bimol: Counting mRNA and Protein Molecules in Single Cells with Digital Microfluidics.

Analyzing single-cell protein and mRNA levels yields invaluable insights into cellular functions and the intricacies of biologically heterogeneous systems. Current joint mRNAs and protein analysis methodologies suffer from relative quantification, low sensitivity, possible background interference, and tedious manual manipulation. Therefore, we propose DMF-Bimol that leverages addressable digital microfluidics to automate digital counting of single-cell mRNA and protein based on proximity ligation assay (PLA) and one-step RT-droplet digital PCR (RT-ddPCR). Through an engineered hydrophilic-hydrophobic interface, DMF-Bimol enables efficient single-cell isolation and lossless protein and nucleic acid processing. The closed droplet reaction system enhances the protein concentration and isolates exogenous contaminants, thereby dramatically improving the efficiency of the PLA reaction. The limit of detection of this approach achieves 3313 protein copies, marking a significant 17-fold enhancement in sensitivity over traditional benchtop PLA. This heightened sensitivity also uncovers a lower correlation between mRNA and protein levels in individual cells (Spearman r = 0.255) than bulk results, reflecting the complex relationship in heterogeneous cells. Using DMF-Bimol, we observed a significant upsurge of CD147 protein in CD138+ myeloma cells but consistent levels of CD147 mRNAs compared with normal leukocytes. This discovery indicates a possible consequence of CD147 oncogenic activation that tends to harness protein translation to bolster tumor cell survival and enhance invasiveness, highlighting the potential of DMF-Bimol in unveiling intricate dynamics in translation processes at the single-cell level.

Comparative Analysis of Gene Expression Analysis Methods for RNA in Situ Hybridization Images

Gene expression analysis is pivotal in cancer research and clinical practice. While traditional methods lack spatial context, RNA in situ hybridization (RNA-ISH) is a powerful technique that retains spatial tissue information. Here, RNAscope score, RT-droplet digital PCR (RT-ddPCR), and automated QuantISH and QuPath were employed for quantifying RNA-ISH expression values from formalin-fixed paraffin-embedded samples. The methods were compared using high-grade serous ovarian carcinoma samples, focusing on CCNE1, WFDC2, and PPIB genes. The findings demonstrate good concordance between automated methods and RNAscope, with RT-ddPCR showing less concordance. Additionally, QuantISH exhibits robust performance, even for low-expressed genes like CCNE1, showcasing its modular design and enhancing accessibility as a viable alternative for gene expression analysis.

Viral interference between severe acute respiratory syndrome coronavirus 2 and influenza A viruses.

Some respiratory viruses can cause a viral interference through the activation of the interferon (IFN) pathway that reduces the replication of another virus. Epidemiological studies of coinfections between SARS-CoV-2 and other respiratory viruses have been hampered by non-pharmacological measures applied to mitigate the spread of SARS-CoV-2 during the COVID-19 pandemic. With the ease of these interventions, SARS-CoV-2 and influenza A viruses can now co-circulate. It is thus of prime importance to characterize their interactions. In this work, we investigated viral interference effects between an Omicron variant and a contemporary influenza A/H3N2 strain, in comparison with an ancestral SARS-CoV-2 strain and the 2009 pandemic influenza A/H1N1 virus. We infected nasal human airway epitheliums with SARS-CoV-2 and influenza, either simultaneously or 24 h apart. Viral load was measured by RT-qPCR and IFN-α/β/λ1/λ2 proteins were quantified by immunoassay. Expression of four interferon-stimulated genes (ISGs; OAS1/IFITM3/ISG15/MxA) was also measured by RT-droplet digital PCR. Additionally, susceptibility of each virus to IFN-α/β/λ2 recombinant proteins was determined. Our results showed that influenza A, and especially A/H3N2, interfered with both SARS-CoV-2 viruses, but that SARS-CoV-2 did not significantly interfere with A/H3N2 or A/H1N1. Consistently with these results, influenza, and particularly the A/H3N2 strain, caused a higher production of IFN proteins and expression of ISGs than SARS-CoV-2. SARS-CoV-2 induced a marginal IFN production and reduced the IFN response during coinfections with influenza. All viruses were susceptible to exogenous IFNs, with the ancestral SARS-CoV-2 and Omicron being less susceptible to type I and type III IFNs, respectively. Thus, influenza A causes a viral interference towards SARS-CoV-2 most likely through an IFN response. The opposite is not necessarily true, and a concurrent infection with both viruses leads to a lower IFN response. Taken together, these results help us to understand how SARS-CoV-2 interacts with another major respiratory pathogen.

Effect of pancreas disease vaccines on infection levels and virus transmission in Atlantic salmon (Salmo salar) challenged with salmonid alphavirus, genotype 2.

Salmonid alphavirus (SAV) causes pancreas disease (PD), which negatively impacts farmed Atlantic salmon. In this study, fish were vaccinated with a DNA-PD vaccine (DNA-PD) and an oil-adjuvanted, inactivated whole virus PD vaccine (Oil-PD). Controls were two non-PD vaccinated groups. Fish were kept in one tank and challenged by cohabitation with SAV genotype 2 in seawater. Protection against infection and mortality was assessed for 84 days (Efficacy study). Nineteen days post challenge (dpc), subgroups of fish from all treatment groups were transferred to separate tanks and cohabited with naïve fish (Transmission study 1) or fish vaccinated with a homologous vaccine (Transmission study 2), to evaluate virus transmission for 26 days (47 dpc). Viremia, heart RT-qPCR and histopathological scoring of key organs affected by PD were used to measure infection levels. RT-droplet digital PCR quantified shedding of SAV into water for transmission studies. The Efficacy study showed that PD associated growth-loss was significantly lower and clearance of SAV2 RNA significantly higher in the PD-DNA group compared to the other groups. The PD-DNA group had milder lesions in the heart and muscle. Cumulative mortality post challenge was low and not different between groups, but the DNA-PD group had delayed time-to-death. In Transmission study 1, the lowest water levels of SAV RNA were measured in the tanks containing the DNA-PD group at 21 and 34 dpc. Despite this, and irrespective of the treatment group, SAV2 was effectively transmitted to the naïve fish during 26-day cohabitation. At 47 dpc, the SAV RNA concentrations in the water were lower in all tanks compared to 34 dpc. In Transmission study 2, none of the DNA-PD immunized cohabitants residing with DNA-PD-vaccinated, pre-challenged fish got infected. In contrast, Oil-PD immunized cohabitants residing with Oil-PD-vaccinated, pre-challenged fish, showed infection levels similar to the naïve cohabitants in Transmission study 1. The results demonstrate that the DNA-PD vaccine may curb the spread of SAV infection as the DNA-PD vaccinated, SAV2 exposed fish, did not spread the infection to cohabiting DNA-PD vaccinated fish. This signifies that herd immunity may be achieved by the DNA-PD vaccine, a valuable tool to control the PD epizootic in farmed Atlantic salmon.

Comparison of Reverse Transcription (RT)-Quantitative PCR and RT-Droplet Digital PCR for Detection of Genomic and Subgenomic SARS-CoV-2 RNA.

Most individuals acutely infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibit mild symptoms. However, 10 to 20% of those infected develop long-term symptoms, referred to as post-coronavirus disease 2019 (COVID-19) condition (PCC). One hypothesis is that PCC might be exacerbated by viral persistence in tissue sanctuaries. Therefore, the accurate detection and quantification of SARS-CoV-2 are not only necessary for viral load monitoring but also crucial for detecting long-term viral persistence and determining whether viral replication is occurring in tissue reservoirs. In this study, the sensitivity and robustness of reverse transcription (RT)-droplet digital PCR (ddPCR) and RT-quantitative PCR (qPCR) techniques have been compared for the detection and quantification of SARS-CoV-2 genomic and subgenomic RNAs from oropharyngeal swabs taken from confirmed SARS-CoV-2-positive, SARS-CoV-2-exposed, and nonexposed individuals as well as from samples from mice infected with SARS-CoV-2. Our data demonstrated that both techniques presented equivalent results in the mid- and high-viral-load ranges. Additionally, RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, allowing the accurate detection of positive results in individuals exposed to the virus. Overall, these data suggest that RT-ddPCR might be an alternative to RT-qPCR for detecting low viral loads in samples and for assessing viral persistence in samples from individuals with PCC. IMPORTANCE We developed one-step reverse transcription (RT)-droplet digital PCR (ddPCR) protocols to detect SARS-CoV-2 RNA and compared them to the gold-standard RT-quantitative PCR (RT-qPCR) method. RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, while both techniques were equivalent in the mid- and high-viral-load ranges. Overall, these results suggest that RT-ddPCR might be a viable alternative to RT-qPCR when it comes to detecting low viral loads in samples, which is a highly relevant issue for determining viral persistence in as-yet-unknown tissue reservoirs in individuals suffering from post-COVID conditions or long COVID.

AKT Isoforms Interplay in High-Grade Serous Ovarian Cancer Prognosis and Characterization.

Simple SummaryNew therapeutical strategies are needed to improve survival in high-grade serous ovarian cancer (HGSOC) patients. AKT inhibitors are promising agents able to act in synergy with PARP inhibitors and platinum-based therapies, but the subset of patients who could benefit from this approach is still unclear. We analyzed AKT isoforms expression in a retrospective cohort and we identified four AKT expression groups related to patients’ survival, tumor morphology and the BRCA status that could help in stratifying patients for future clinical trials.High-grade serous ovarian cancer (HGSOC) is among the deadliest gynecological malignancies. The acquired resistance to platinum-based therapies and the intrinsic heterogeneity of the disease contribute to the low survival rate. To improve patients’ outcomes, new combinatorial approaches able to target different tumor vulnerabilities and enhance the efficacy of the current therapies are required. AKT inhibitors are promising antineoplastic agents able to act in synergy with PARP inhibitors, but the spectrum of patients who can benefit from this combination is unclear, since the role of the three different isoforms of AKT is still unknown. Here, we study the expression of AKT isoforms on a retrospective cohort of archive tissue by RT-droplet digital PCR (ddPCR) analyzing their association with the clinicopathological features of patients. Based on AKT1/AKT2 and AKT1/AKT3 ratios, we define four AKT classes which were related to patients’ survival, tumor morphology and BRCA1 expression. Moreover, our results show that high AKT3 expression levels were frequently associated with tumors having classic features, a low number of mitoses and the presence of psammoma bodies. Overall, our study obtains new insights on AKT isoforms and their associations with the clinicopathological features of HGSOC patients. These evidences could help to better define the subsets of patients who can benefit from AKT and PARP inhibitors therapy in future clinical trials.

Hsa_circ_0001275 Is One of a Number of circRNAs Dysregulated in Enzalutamide Resistant Prostate Cancer and Confers Enzalutamide Resistance In Vitro

Simple SummaryAlthough newer generations of androgen deprivation therapy such as enzalutamide are providing hope, it is clinically challenging to deliver effective therapy to individuals with metastatic castrate-resistant prostate cancer. Between 20–40% of patients have intrinsic resistance to therapy and all patients will ultimately experience disease progression due to acquired resistance, which is a significant clinical dilemma. The aim of our study was to evaluate the role of circular RNAs (circRNAs) in enzalutamide-resistant prostate cancer as part of the effort to identify useful biomarkers for patient selection and potential new therapeutic targets. We confirmed that hsa_circ_0001275 was highly upregulated in an enzalutamide resistant cell line and demonstrated that its overexpression resulted in increased enzalutamide resistance. Our data showed that hsa_circ_0001275 was not expressed abundantly in patient plasma samples, however, a trend of expression was evident which paralleled disease activity indicating a possible association with enzalutamide resistance. Overall, we have provided evidence that hsa_circ_0001275 promotes enzalutamide resistance and thus may serve as a potential therapeutic target.Background: Enzalutamide is part of the treatment regimen for metastatic castration-resistant prostate cancer (MCRPC). However, both intrinsic and acquired resistance to the drug remain substantial clinical quandaries. circRNAs, a novel type of non-coding RNA, have been identified in a number of cancers including prostate cancer and have been associated with cancer development and progression. circRNAs have shown great potential as clinically useful blood-based ‘liquid biopsies’ and as therapeutic targets in prostate cancer. The aim of this study was to examine the role of circRNA transcripts in enzalutamide-resistant prostate cancer cells and assess their utility as biomarkers. Methods: An isogenic cell line model of enzalutamide resistance was subjected to circRNA microarray profiling. Several differentially expressed circRNAs, along with their putative parental genes were validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). circRNAs of interest were stably overexpressed in the control cell line and drug sensitivity was assessed using an ELISA-based proliferation assay. The candidate circRNA, hsa_circ_0001275, was measured in patient plasma samples using RT-droplet digital PCR (RT-ddPCR). Results: hsa_circ_0001275 and its parental gene, PLCL2, were significantly up-regulated in strongly resistant clones vs. control (p < 0.05). Overexpression of hsa_circ_0001275 in the control cell line resulted in increased resistance to enzalutamide (p < 0.05). While RT-ddPCR analysis of hsa_circ_0001275 expression in plasma samples of 44 clinical trial participants showed a trend that mirrored the stages of disease activity (as defined by PSA level), the association did not reach statistical significance. Conclusions: Our data suggest that increased levels of hsa_circ_0001275 contribute to enzalutamide resistance. hsa_circ_0001275 plasma expression showed a trend that mirrors the PSA level at specific disease time points, indicating that circRNAs mirror disease recurrence and burden and may be associated with enzalutamide resistance.

CRED9: A differentially expressed elncRNA regulates expression of transcription factor CEBPA.

Enhancer RNAs (eRNA) are noncoding transcripts produced from active enhancers and have potential gene regulatory function. CCAAT enhancer-binding protein alpha (CEBPA) is a transcription factor generally involved in metabolism, cell cycle inhibition, hematopoiesis, adipogenesis, hepatogenesis, and is associated with tumorigenesis. In this study, we demonstrate that an enhancer-associated long noncoding RNA (elncRNA), transcribed from an enhancer located 9 kb downstream from the transcriptional start site (TSS) of CEBPA, positively regulates the expression of CEBPA. As a result, we named this elncRNA “CEBPA regulatory elncRNA downstream 9 kb” or “CRED9.” CRED9 expression level positively correlates with CEBPA mRNA expression across multiple cell lines as detected by RT droplet digital PCR. Knockdown of CRED9 resulted in a reduction of CEBPA mRNA expression in Hep3B cells. Additionally, CRED9 knockdown in Hep3B and HepG2 cells resulted in lower CEBPA protein expression. We also found that knockdown of CRED9 in Hep3B cells caused a 57.8% reduction in H3K27ac levels at the +9 kb CEBPA enhancer. H3K27ac has previously been described as a marker of active enhancers. Taken together, the evidence presented here supports a previously proposed model whereby, in some contexts, eRNA transcripts are necessary to amplify and maintain H3K27ac levels at a given enhancer. Ultimately, this study adds to the growing body of evidence that elncRNA transcripts have important roles in enhancer function and gene regulation.

Application of One-Step Reverse Transcription Droplet Digital PCR for Dengue Virus Detection and Quantification in Clinical Specimens

Detection and quantification of viruses in laboratory and clinical samples are standard assays in dengue virus (DENV) studies. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) is considered to be the standard for DENV detection and quantification due to its high sensitivity. However, qRT-PCR offers only quantification relative to a standard curve and consists of several “in-house” components resulting in interlaboratory variations. We developed and optimized a protocol for applying one-step RT-droplet digital PCR (RT-ddPCR) for DENV detection and quantification. The lower limit of detection (LLOD95) and the lower limit of quantification (LLOQ) for RT-ddPCR were estimated to be 1.851 log10-copies/reaction and 2.337 log10-copies/reaction, respectively. The sensitivity of RT-ddPCR was found to be superior to qRT-PCR (94.87% vs. 90.38%, p = 0.039) while no false positives were detected. Quantification of DENV in clinical samples was independently performed in three laboratories showing interlaboratory variations with biases <0.5 log10-copies/mL. The RT-ddPCR protocol presented here could help harmonize DENV quantification results and improve findings in the field such as identifying a DENV titer threshold correlating with disease severity.

Small Interfering RNAs Are Highly Effective Inhibitors of Crimean-Congo Hemorrhagic Fever Virus Replication In Vitro.

Crimean-Congo hemorrhagic fever virus (CCHFV) is one of the prioritized diseases of the World Health Organization, considering its potential to create a public health emergency and, more importantly, the absence of efficacious drugs and/or vaccines for treatment. The highly pathogenic characteristic of CCHFV restricts research to BSL-4 laboratories, which complicates effective research and developmental strategies. In consideration of antiviral therapies, RNA interference can be used to suppress viral replication by targeting viral genes. RNA interference uses small interfering RNAs (siRNAs) to silence genes. The aim of our study was to design and test siRNAs in vitro that inhibit CCHFV replication and can serve as a basis for further antiviral therapies. A549 cells were infected with CCHFV after transfection with the siRNAs. Following 72 h, nucleic acid from the supernatant was extracted for RT Droplet Digital PCR analysis. Among the investigated siRNAs we identified effective candidates against all three segments of the CCHF genome. Consequently, blocking any segment of CCHFV leads to changes in the virus copy number that indicates an antiviral effect of the siRNAs. In summary, we demonstrated the ability of specific siRNAs to inhibit CCHFV replication in vitro. This promising result can be integrated into future anti-CCHFV therapy developments.

A fast, efficient and high-throughput procedure involving laser microdissection and RT droplet digital PCR for tissue-specific expression profiling of rice roots

BackgroundIn rice, the cortex and outer tissues play a key role in submergence tolerance. The cortex differentiates into aerenchyma, which are air-containing cavities that allow the flow of oxygen from shoots to roots, whereas exodermis suberification and sclerenchyma lignification limit oxygen loss from the mature parts of roots by forming a barrier to root oxygen loss (ROL). The genes and their networks involved in the cellular identity and differentiation of these tissues remain poorly understood. Identification and characterization of key regulators of aerenchyma and ROL barrier formation require determination of the specific expression profiles of these tissues.ResultsWe optimized an approach combining laser microdissection (LM) and droplet digital RT-PCR (ddRT-PCR) for high-throughput identification of tissue-specific expression profiles. The developed protocol enables rapid (within 3 days) extraction of high-quality RNA from root tissues with a low contamination rate. We also demonstrated the possibility of extracting RNAs from paraffin blocks stored at 4 °C without any loss of quality. We included a detailed troubleshooting guide that should allow future users to adapt the proposed protocol to other tissues and/or species. We demonstrated that our protocol, which combines LM with ddRT-PCR, can be used as a complementary tool to in situ hybridization for tissue-specific characterization of gene expression even with a low RNA concentration input. We illustrated the efficiency of the proposed approach by validating three of four potential tissue-specific candidate genes detailed in the RiceXpro database.ConclusionThe detailed protocol and the critical steps required to optimize its use for other species will democratize tissue-specific transcriptome approaches combining LM with ddRT-PCR for analyses of plants.

Dynamics of Blood Viral Load Is Strongly Associated with Clinical Outcomes in Coronavirus Disease 2019 (COVID-19) Patients: A Prospective Cohort Study

The prevalence and clinical relevance of viremia in patients with coronavirus disease 2019 (COVID-19) have not been well studied. A prospective cohort study was designed to investigate blood viral load and clearance kinetics in 52 patients (median age, 62 years; 31 [59.6%] male) and explore their association with clinical features and outcomes based on a novel one-step RT droplet digital PCR (RT-ddPCR). By using one-step RT-ddPCR, 92.3% (48 of 52) of this cohort was quantitatively detected with viremia. The concordance between the blood and oropharyngeal swab tests was 60.92% (53 of 87). One-step RT-ddPCR was tested with a 3.03% false-positive rate and lower 50% confidence interval of detection at 54.026 copies/mL plasma. There was no reduction in the blood viral load in all critical patients, whereas the general and severe patients exhibited a similar ability to clear the viral load. The viral loads in critical patients were significantly higher than those in their general and severe counterparts. Among the 52 study patients, 30 (58%) were discharged from the hospital. Among half of the 30 discharged patients, blood viral load remained positive, of which 76.9% (10 of 13) completely cleared their blood viral load at follow-up. Meanwhile, none of their close contacts had evidence of infection. Quantitative determination of the blood viral test is of great clinical significance in the management of patients with coronavirus disease 2019.

One-step duplex RT-droplet digital PCR assay for the detection of norovirus GI and GII in lettuce and strawberry

The study was designed to develop a sensitive one-step duplex reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) to detect norovirus genogroup I and II (NoV GI and GII) in lettuce and strawberry. The specificity, sensitivity, repeatability and robustness of the assay was compared with RT-qPCR. The lowest concentration detected by RT-ddPCR for NoV GI and NoV GII were 4.68 and 8.47 copies/μL respectively, much lower than that of RT-qPCR with a number of 46.8 and 84.7 copies/μL, respectively. Lettuce and strawberry samples were artificially contaminated with NoV GI and GII suspensions, with inoculum size of 3.00 × 106 to 1.70 × 108 copies and 4.80 × 105 to 2.50 × 107 copies, respectively. Strawberry spiked with low inoculum size revealed positive results by RT-ddPCR, while recorded negative by RT-qPCR. Meanwhile, RT-ddPCR also showed a higher average recovery rate for NoV in lettuce and strawberry than RT-qPCR.The limit of detection (LoDs) of RT-ddPCR for NoVs in lettuce was 2.32 × 104 copies/25g (NoV GI) and 2.36 × 104 ciopies/25g (NoV GII), and that in strawberry was 2.56 × 104 copies/25g (NoV GI) and 2.64 × 104 ciopies/25g (NoV GII), which were 10 folds lower than that of RT-qPCR. The developed duplex RT-ddPCR assay exhibited stability and increased capacity to resist inhibitors in food samples with low concentration of NoV, making it a reliable method to avoid false negative result as opposed to RT-qPCR. In conclusion, one-step RT-ddPCR method developed in this study is pertinent in detecting foodborne virus such as NoV.

Detection of SARS-CoV-2 RNA in commercial passenger aircraft and cruise ship wastewater: a surveillance tool for assessing the presence of COVID-19 infected travellers.

BackgroundWastewater-based epidemiology (WBE) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be an important source of information for coronavirus disease 2019 (COVID-19) management during and after the pandemic. Currently, governments and transportation industries around the world are developing strategies to minimize SARS-CoV-2 transmission associated with resuming activity. This study investigated the possible use of SARS-CoV-2 RNA wastewater surveillance from airline and cruise ship sanitation systems and its potential use as a COVID-19 public health management tool.MethodsAircraft and cruise ship wastewater samples (n = 21) were tested for SARS-CoV-2 using two virus concentration methods, adsorption–extraction by electronegative membrane (n = 13) and ultrafiltration by Amicon (n = 8), and five assays using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and RT-droplet digital PCR (RT-ddPCR). Representative qPCR amplicons from positive samples were sequenced to confirm assay specificity.ResultsSARS-CoV-2 RNA was detected in samples from both aircraft and cruise ship wastewater; however concentrations were near the assay limit of detection. The analysis of multiple replicate samples and use of multiple RT-qPCR and/or RT-ddPCR assays increased detection sensitivity and minimized false-negative results. Representative qPCR amplicons were confirmed for the correct PCR product by sequencing. However, differences in sensitivity were observed among molecular assays and concentration methods.ConclusionsThe study indicates that surveillance of wastewater from large transport vessels with their own sanitation systems has potential as a complementary data source to prioritize clinical testing and contact tracing among disembarking passengers. Importantly, sampling methods and molecular assays must be further optimized to maximize detection sensitivity. The potential for false negatives by both wastewater testing and clinical swab testing suggests that the two strategies could be employed together to maximize the probability of detecting SARS-CoV-2 infections amongst passengers.

Dysregulation of apoptosis and autophagy gene expression in peripheral blood mononuclear cells of efficiently treated HIV-infected patients.

We measure the transcript levels of the proapoptotic GALIG, antiapoptotic MCL1 genes and those of the autophagy genes BECN1, MAP1LC3B, ATG9a, P62/SQSTM1, GABARAP, GABARAPL1 and GABARAPL2 to define if mRNA alteration can characterize HIV-infected patients effectively treated with combined antiretroviral therapy (cART). Monocentric pilot study conducted on peripheral blood mononuclear cell (PBMC) of 40 uninfected donors and 27 HIV-positive patients effectively treated by cART for at least 8.4 years. Transcripts of the various genes were quantified by reverse transcription (RT)-quantitative PCR (qPCR) and RT-droplet digital PCR and compared using the standard statistical Mann-Whitney U test and machine learning algorithms. A concomitant overexpression of GALIG and MCL1 is detected in PBMC of effectively cART-treated patients. Overexpression of MAP1LC3B and GABARAPL1 is also measured, whereas BECN1 is underexpressed. Finally, accurate classification (94.5%) of our PBMC samples as HIV-negative donors or HIV-positive cART-treated is obtained in three separate machine-learning algorithms with GABARAPL1 and ATG9a as input variables. cART-treated HIV patients display altered transcript levels for three genes of basal autophagy. Some of these alterations may appear contradictory: BECN1 and ATG9a, both key actors in the formation of mammalian autophagosome, exhibit decreased amount of transcripts, whereas mRNA from the ATG8 family increase. Given the known role of impaired basal autophagy in immune senescence and chronic inflammation, the functional significance of our findings should be explored in larger studies.

Comparison between RT droplet digital PCR and RT real-time PCR for quantification of noroviruses in oysters

Oysters are frequently associated with norovirus outbreaks, but the presence of norovirus RNA in oysters does not necessarily imply a health risk to humans. There is a close link between human illness and consumption of oysters with high levels of norovirus RNA, but oysters with low levels of norovirus RNA are more unlikely to be associated with illness. Reliable and precise quantification methods are therefore important for outbreak investigations and risk assessments. This study optimised and validated RT droplet digital PCR (RT-ddPCR) assays for quantification of norovirus genogroups I and II in artificially contaminated oysters, and compared them with the standard method, RT real-time PCR (RT-qPCR). The two methods had comparable 95% limits of detection, but RT-ddPCR generally showed greater precision in quantification. Differences between fluorometric measurements and quantification with RT-ddPCR were determined on in vitro transcribed RNA with targets for norovirus genogroups I and II. Quantification by RT-ddPCR was on average 100 times lower than the fluorometric value for norovirus GI and 15.8 times lower than the fluorometric value for norovirus GII. The large inter-assay difference observed highlights the need for monitoring the RT efficiency in RT-ddPCR, especially when results from different assays are compared. Overall, this study suggests that RT-ddPCR can be a suitable method for precise quantification of norovirus genogroups I and II in oysters.

Evaluation of relative quantification of alternatively spliced transcripts using droplet digital PCR

IntroductionFor the relative quantification of isoform expression, RT-qPCR has been the gold standard for over a decade. More recently, digital PCR is becoming widely implemented, as it is promised to be more accurate, sensitive and less affected by inhibitors, without the need for standard curves. In this study we evaluated RT-qPCR versus RT-droplet digital PCR (ddPCR) for the relative quantification of isoforms in controls and carriers of the splice site mutation BRCA1 c.212+3A>G, associated with increased expression of several isoforms. Materials and methodsRNA was extracted from EBV cell lines of controls and heterozygous BRCA1 c.212+3A>G carriers. Transcript-specific plasmids were available to determine the efficiency, precision, reproducibility and accuracy of each method. ResultsBoth ddPCR and RT-qPCR were able to accurately quantify all targets and showed the same LOB, LOD and LOQ; also precision and reproducibility were similar. Both techniques have the same dynamic range and linearity at biologically relevant template concentrations. However, a significantly higher cost and workload was required for ddPCR experiments. ConclusionsOur study recognizes the potential and validity of digital PCR but shows the value of a highly optimized qPCR for the relative quantification of isoforms. Cost efficiency and simplicity turned out to be better for RT-qPCR.

One-step RT-droplet digital PCR: a breakthrough in the quantification of waterborne RNA viruses

Water contamination by viruses has an increasing worldwide impact on human health, and has led to requirements for accurate and quantitative molecular tools. Here, we report the first one-step reverse-transcription droplet digital PCR-based absolute quantification of a RNA virus (rotavirus) in different types of surface water samples. This quantification method proved to be more precise and more tolerant to inhibitory substances than the benchmarking reverse-transcription real-time PCR (RT-qPCR), and needs no standard curve. This new tool is fully amenable for the quantification of viruses in the particularly low concentrations usually found in water samples.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-013-7476-y) contains supplementary material, which is available to authorized users.

O.118 Management of patients with bisphosphonates-induced osteonecrosis

For the relative quantification of isoform expression, RT-qPCR has been the gold standard for over a decade. More recently, digital PCR is becoming widely implemented, as it is promised to be more accurate, sensitive and less affected by inhibitors, without the need for standard curves. In this study we evaluated RT-qPCR versus RT-droplet digital PCR (ddPCR) for the relative quantification of isoforms in controls and carriers of the splice site mutation BRCA1 c.212+3A>G, associated with increased expression of several isoforms.RNA was extracted from EBV cell lines of controls and heterozygous BRCA1 c.212+3A>G carriers. Transcript-specific plasmids were available to determine the efficiency, precision, reproducibility and accuracy of each method.Both ddPCR and RT-qPCR were able to accurately quantify all targets and showed the same LOB, LOD and LOQ; also precision and reproducibility were similar. Both techniques have the same dynamic range and linearity at biologically relevant template concentrations. However, a significantly higher cost and workload was required for ddPCR experiments.Our study recognizes the potential and validity of digital PCR but shows the value of a highly optimized qPCR for the relative quantification of isoforms. Cost efficiency and simplicity turned out to be better for RT-qPCR.