Identifying transcriptional miRNA biomarkers by integrating high-throughput sequencing and real-time PCR data

Methods ◽  
2013 ◽  
Vol 59 (1) ◽  
pp. 154-163 ◽  
Author(s):  
Sven Rahmann ◽  
Marcel Martin ◽  
Johannes H. Schulte ◽  
Johannes Köster ◽  
Tobias Marschall ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
High Throughput Sequencing

scholarly journals Verification of miRNAs in ginseng decoction by high-throughput sequencing and quantitative real-time PCR

Heliyon ◽  
2019 ◽  
Vol 5 (4) ◽  
pp. e01418 ◽  
Author(s):  
Yingfang Wang ◽  
Mengyuan Peng ◽  
Wenjuan Wang ◽  
Yanlin Chen ◽  
Zhihua He ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
High Throughput Sequencing ◽  
Quantitative Real Time Pcr

scholarly journals Diversity of KIR genes and their HLA-C ligands in Ugandan populations with historically varied malaria transmission intensity

2021 ◽  
Author(s):  
Stephen Tukwasibwe ◽  
James A. Traherne ◽  
Olympe Chazara ◽  
Jyothi Jayaraman ◽  
John Trowsdale ◽  
...  
Keyword(s):  
Infectious Diseases ◽  
Malaria Transmission ◽  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Association Studies ◽  
Transmission Intensity ◽  
Malaria Transmission Intensity ◽  
Significant Difference ◽  
Pcr Method

Abstract Background: Malaria is one of the most serious infectious diseases in the world. The malaria burden is greatly affected by human immunity, and immune responses vary between populations. Genetic diversity in KIR and HLA-C genes, which are important in immunity to infectious diseases, is likely to play a role in this heterogeneity. Several studies have shown that KIR and HLA-C genes influence the immune response to viral infections, but few studies have examined the role of KIR and HLA-C in malaria infection, and these have used low-resolution genotyping. The aim of this study was to determine whether genetic variation in KIR and their HLA-C ligands differ in Ugandan populations with historically varied malaria transmission intensity using more comprehensive genotyping approaches.Methods: High throughput multiplex quantitative real-time PCR method was used to genotype KIR genetic variants and copy number variation and a high-throughput real-time PCR method was developed to genotype HLA-C1 and C2 allotypes for 1,344 participants, aged 6 months to 10 years, enrolled from Ugandan populations with historically high (Tororo District), medium (Jinja District) and low (Kanungu District) malaria transmission intensity. Results: The prevalence of KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes was significantly lower in populations from Kanungu compared to Tororo (7.6% vs. 13.2%: p=0.006, 57.2% vs. 66.4%: p=0.005, 33.2% vs. 46.6%: p<0.001 and 19.7% vs. 26.7%: p=0.014 respectively) or Jinja (7.6% vs.18.1%: p<0.001, 57.2% vs. 63.8%: p=0.048, 33.2% vs. 43.5%: p=0.002 and 19.7% vs. 30.4%: p<0.001 respectively). The prevalence of homozygous HLA-C2 was significantly higher in populations from Kanungu (31.6%) compared to Jinja (21.4%), p=0.043, with no significant difference between Kanungu and Tororo (26.7%), p=0.296. Conclusions: The KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes may partly explain differences in transmission intensity of malaria since these genes have been positively selected for in places with historically high malaria transmission intensity. The high-throughput multiplex real-time HLA-C genotyping PCR method developed will be useful in disease association studies involving large cohorts.

scholarly journals Estimating Clinically Relevant Cut-Off Values for a High-Throughput Quantitative Real-Time PCR Detecting Bacterial Respiratory Pathogens in Cattle

2021 ◽  
Vol 8 ◽  
Author(s):  
Alicia F. Klompmaker ◽  
Maria Brydensholt ◽  
Anne Marie Michelsen ◽  
Matthew J. Denwood ◽  
Carsten T. Kirkeby ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Pasteurella Multocida ◽  
Age Groups ◽  
Therapeutic Interventions ◽  
Respiratory Pathogens ◽  
Logistic Regression Models ◽  
Histophilus Somni ◽  
Nasal Swabs

Bovine respiratory disease (BRD) results from interactions between pathogens, environmental stressors, and host factors. Obtaining a diagnosis of the causal pathogens is challenging but the use of high-throughput real-time PCR (rtPCR) may help target preventive and therapeutic interventions. The aim of this study was to improve the interpretation of rtPCR results by analysing their associations with clinical observations. The objective was to develop and illustrate a field-data driven statistical method to guide the selection of relevant quantification cycle cut-off values for pathogens associated with BRD for the high-throughput rtPCR system “Fluidigm BioMark HD” based on nasal swabs from calves. We used data from 36 herds enrolled in a Danish field study where 340 calves within pre-determined age-groups were subject to clinical examination and nasal swabs up to four times. The samples were analysed with the rtPCR system. Each of the 1,025 observation units were classified as sick with BRD or healthy, based on clinical scores. The optimal rtPCR results to predict BRD were investigated for Pasteurella multocida, Mycoplasma bovis, Histophilus somni, Mannheimia haemolytica, and Trueperella pyogenes by interpreting scatterplots and results of mixed effects logistic regression models. The clinically relevant rtPCR cut-off suggested for P. multocida and M. bovis was ≤ 21.3. For H. somni it was ≤ 17.4, while no cut-off could be determined for M. haemolytica and T. pyogenes. The demonstrated approach can provide objective support in the choice of clinically relevant cut-offs. However, for robust performance of the regression model sufficient amounts of suitable data are required.

scholarly journals Massively parallel single-molecule telomere length measurement with digital real-time PCR

2020 ◽  
Vol 6 (34) ◽  
pp. eabb7944 ◽  
Author(s):  
Yongqiang Luo ◽  
Ramya Viswanathan ◽  
Manoor Prakash Hande ◽  
Amos Hong Pheng Loh ◽  
Lih Feng Cheow
Keyword(s):  
Telomere Length ◽  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Real Time Pcr ◽  
Length Distribution ◽  
Telomere Maintenance ◽  
Massively Parallel ◽  
Primary Tumors ◽  
Absolute Length

Telomere length is a promising biomarker for age-associated diseases and cancer, but there are still substantial challenges to routine telomere analysis in clinics because of the lack of a simple and rapid yet scalable method for measurement. We developed the single telomere absolute-length rapid (STAR) assay, a novel high-throughput digital real-time PCR approach for rapidly measuring the absolute lengths and quantities of individual telomere molecules. We show that this technique provides the accuracy and sensitivity to uncover associations between telomere length distribution and telomere maintenance mechanisms in cancer cell lines and primary tumors. The results indicate that the STAR assay is a powerful tool to enable the use of telomere length distribution as a biomarker in disease and population-wide studies.

scholarly journals Development of a High-Throughput Quantitative Assay for Detecting Herpes Simplex Virus DNA in Clinical Samples

1999 ◽  
Vol 37 (6) ◽  
pp. 1941-1947 ◽  
Author(s):  
Alexander J. Ryncarz ◽  
James Goddard ◽  
Anna Wald ◽  
Meei-Li Huang ◽  
Bernard Roizman ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Genital Tract ◽  
Clinical Samples ◽  
Pcr Assay ◽  
The Mean ◽  
Simplex Virus

We have developed a high-throughput, semiautomated, quantitative fluorescence-based PCR assay to detect and type herpes simplex virus (HSV) DNA in clinical samples. The detection assay, which uses primers to the type-common region of HSV glycoprotein B (gB), was linear from <10 to 108 copies of HSV DNA/20 μl of sample. Among duplicate samples in reproducibility runs, the assay showed less than 5% variability. We compared the fluorescence-based PCR assay with culture and gel-based liquid hybridization system with 335 genital tract specimens from HSV type 2 (HSV-2)-seropositive persons attending a research clinic and 380 consecutive cerebrospinal fluid (CSF) samples submitted to a diagnostic virology laboratory. Among the 162 culture-positive genital tract specimens, TaqMan PCR was positive for 157 (97%) specimens, whereas the quantitative-competitive PCR was positive for 144 (89%) specimens. Comparisons of the mean titer of HSV DNA detected by the two assays revealed that the mean titer detected by the gel-based system was slightly higher (median, 1 log). These differences in titers were in part related to the fivefold difference in the amount of HSV DNA used in the amplicon standards with the two assays. Among the 380 CSF samples, 42 were positive by both assays, 13 were positive only by the assay with the agarose gel, and 3 were positive only by the assay with the fluorescent probe. To define the subtype of HSV DNA detected in the screening assay, we also designed one set of primers which amplifies the gG regions of both types of HSV and probes which are specific to either HSV-1 (gG1) or HSV-2 (gG2). These probes were labeled with different fluorescent dyes (6-carboxyfluorescein for gG2 and 6-hexachlorofluorescein for gG1) to enable detection in a single PCR. In mixing experiments the probes discriminated the correct subtype in mixtures with up to a 7-log-higher concentration of the opposite subtype. The PCR typing results showed 100% concordance with the results obtained by assays with monoclonal antibodies against HSV-1 or HSV-2. Thus, while the real-time PCR is slightly less sensitive than the gel-based liquid hybridization system, the high throughput, the lack of contamination during processing, the better reproducibility, and the better ability to type the isolates rapidly make the real-time PCR a valuable tool for clinical investigation and diagnosis of HSV infection.

scholarly journals Validation of a real-time PCR assay for high-throughput detection of Avibacterium paragallinarum in chicken respiratory sites

2019 ◽  
Vol 31 (5) ◽  
pp. 714-718 ◽  
Author(s):  
Kristin A. Clothier ◽  
Simone Stoute ◽  
Andrea Torain ◽  
Beate Crossley
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Bacterial Flora ◽  
Clinical Samples ◽  
Single Tube ◽  
Avibacterium Paragallinarum ◽  
Normal Bacterial Flora ◽  
Close Relatives

Avibacterium paragallinarum is the causative agent of infectious coryza, a highly contagious respiratory disease in chickens. Given its fastidious nature, this bacterium is difficult to recover and identify, particularly from locations colonized by normal bacterial flora. Standard PCR methods have been utilized for detection but are labor-intensive and not feasible for high-throughput testing. We evaluated a real-time PCR (rtPCR) method targeting the HPG-2 region of A. paragallinarum, and validated a high-throughput extraction for this assay. Using single-tube extraction, the rtPCR detected 4 A. paragallinarum (ATCC 29545T and 3 clinical) isolates with a limit of detection (LOD) of 10 cfu/mL and a PCR efficiency of 89–111%. Cross-reaction was not detected with 33 non– A. paragallinarum, all close relatives from the family Pasteurellaceae. Real-time PCR testing on extracts of 66 clinical samples (choana, sinus, or trachea) yielded 98.2% (35 of 36 on positives, 30 of 30 on negatives) agreement with conventional PCR. Duplicate samples tested in a 96-well format extraction in parallel with the single-tube method produced equivalent LOD on all A. paragallinarum isolates, and 96.8% agreement on 93 additional clinical samples extracted with both procedures. This A. paragallinarum rtPCR can be utilized for outbreak investigations and routine monitoring of susceptible flocks.

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