Journal: Parasitology

Article Title: Quantitative monitoring of experimental and human leishmaniasis employing amastigote-specific genes

doi: 10.1017/S0031182022000610

Figure Lengend Snippet: Status of A2 and amastin in hepatic and splenic tissues of infected Syrian golden hamsters. (A, B. i–ii) Representative 1-dimensional plots of droplets measured for fluorescence signals (amplitude indicated on y-axis) emitted from A2 (A) and amastin (B) in infected (n = 10) and non-infected (n = 10) liver and splenic tissues of hamsters. Lane 1: negative controls; lane 2: non-template controls; lanes 3–4: non-infected liver and non-infected splenic tissues; lanes 5–7: infected hepatic (i) and splenic tissues (ii) respectively. EvaGreen-bound positive droplets are shown in blue while negative droplets are shown in black, with expression of the genes quantified as copies per μL. (A, B. iii, v) Bar graphs showing mRNA expression of A2 (A) and amastin (B) in infected hepatic (iii) and splenic tissues (v) of hamsters. Each horizontal bar represents mean ± s.e.m. of 10 animals in duplicates. #P < 0.001 as compared to uninfected counterparts. (A, B. iv, vi) Correlation between no. of parasites per μg gDNA with expression of A2 (A) and amastin (B) in infected liver tissues (iv) and splenic tissues (vi) respectively; (C) receiver operating characteristic (ROC) curve for A2 and amastin in hamster liver (i, iii) and splenic (ii, iv) tissues. Linear regression between predicted and actual ddPCR output (copies per μL) was observed for all tested targets and measured as R2 in hamster liver (v, vii) and splenic (vi, viii) tissues.

Article Snippet: Briefly, the optimized reaction mixture containing cDNA (50 ng) was added to a ddPCR EvaGreen Supermix (Bio-Rad Laboratories, CA, USA) containing 100 n m mL −1 of each forward and reverse primer, final volume being 20 μ L. For negative controls (NC), 10 μ L nuclease-free water was added along with 10 μ L of ddPCR Eva Green Super mix, and in case of non-template control (NTC), nuclease-free water was used instead of cDNA.

Techniques: Infection, Fluorescence, Expressing

Journal: Parasitology

Article Title: Quantitative monitoring of experimental and human leishmaniasis employing amastigote-specific genes

doi: 10.1017/S0031182022000610

Figure Lengend Snippet: Status of A2 (A) and amastin (B) in L. donovani-infected murine peritoneal macrophages. (A, B. i, iv) Representative 1-dimensional plots of droplets measured for fluorescence signals (amplitude indicated on y-axis) emitted from A2 (A) and amastin (B) in murine peritoneal macrophages infected with L. donovani followed by treatment with HePC (i) or AmphoB (iv) as described in Materials and methods section. Lane 1: negative control, lane 2: non-template control, lanes 3, 4: log phase and stationary phase promastigotes respectively, lane 5: control, non-infected peritoneal macrophages, lane 6: infected peritoneal macrophages, lanes 7–11: HePC (0.3–5 μm) [i] and AmphoB (3.12–50 nm) [iv] treated peritoneal macrophages. EvaGreen-bound positive droplets are shown in blue while negative droplets are shown in black, with expression of the genes quantified as the copies per μL. (A, B. ii, v) Bar graphs showing mRNA expression of A2 (A) and amastin (B) in peritoneal macrophages infected with L. donovani and treated with HePC (ii) or Ampho B (v). Each horizontal bar represents mean ± s.e.m. of at least 3 different experiments in duplicates; *P < 0.05, **P < 0.01 and ***P < 0.001 as compared to infected macrophages. (A, B. iii, vi) Anti-amastigote activities of HePC (iii) and AmphoB (vi) were evaluated as described in Materials and methods section. (C) Receiver operating characteristic (ROC) curve for A2 (i) and amastin (iii) in infected peritoneal macrophages. Linear regression between predicted and actual ddPCR output (copies per μL) was observed for A2 (ii) and amastin (iv). (D) Representative images showing the anti-amastigote activity of HePC and AmphoB in AG83-infected peritoneal macrophages. Murine peritoneal macrophages (i) infected with L. donovani promastigotes (ii) treated with 0.6 μm HePC (iii) or 12.5 nm AmphoB (iv); Bar-10 μm, magnification 1000×.

Article Snippet: Briefly, the optimized reaction mixture containing cDNA (50 ng) was added to a ddPCR EvaGreen Supermix (Bio-Rad Laboratories, CA, USA) containing 100 n m mL −1 of each forward and reverse primer, final volume being 20 μ L. For negative controls (NC), 10 μ L nuclease-free water was added along with 10 μ L of ddPCR Eva Green Super mix, and in case of non-template control (NTC), nuclease-free water was used instead of cDNA.

Techniques: Infection, Fluorescence, Negative Control, Expressing, Activity Assay

Journal: Parasitology

Article Title: Quantitative monitoring of experimental and human leishmaniasis employing amastigote-specific genes

doi: 10.1017/S0031182022000610

Figure Lengend Snippet: Status of amastigote-specific genes A2 (A) and amastin (B) in PKDL. (A, B) Representative 1-dimensional plots of droplets measured for fluorescence signals (amplitude indicated on y-axis) emitted from A2 (A) and amastin (B) in skin biopsies sourced from PKDL cases (n = 10) and healthy controls (n = 10). EvaGreen-bound positive droplets are shown in blue while negative droplets are shown in black, with expression of genes quantified as copies per μL. (i) Lane 1: negative controls, lane 2: non-template controls, lanes 3–5: healthy controls for A2 (A) and Amastin (B) respectively. (ii) Lane 1: negative controls, lane 2: non-template controls, lanes 3–12: PKDL cases tested for expression of A2 (A) and Amastin (B) respectively. (iii) Violin plots showing mRNA expression of A2 (A) and amastin (B) in PKDL cases and healthy controls (n = 10). Each horizontal bar represents mean ± s.e.m. of 10 individuals in duplicates; @P < 0.001 as compared to healthy controls. (iv) Correlation between number of parasites per μg gDNA with A2 (A) and amastin (B) in PKDL cases. (C) Receiver operating characteristic (ROC) curve for A2 (i) and amastin (iii) in PKDL cases. Linear regression between predicted and actual ddPCR output (copies per μL) was observed for all tested targets in PKDL for A2 (ii) and amastin (iv).

Article Snippet: Briefly, the optimized reaction mixture containing cDNA (50 ng) was added to a ddPCR EvaGreen Supermix (Bio-Rad Laboratories, CA, USA) containing 100 n m mL −1 of each forward and reverse primer, final volume being 20 μ L. For negative controls (NC), 10 μ L nuclease-free water was added along with 10 μ L of ddPCR Eva Green Super mix, and in case of non-template control (NTC), nuclease-free water was used instead of cDNA.

Techniques: Fluorescence, Expressing

Journal: Current protocols in human genetics

Article Title: Droplet Digital PCR with EvaGreen Assay: Confirmational Analysis of Structural Variants

doi: 10.1002/cphg.58

Figure Lengend Snippet: Overview of the ddPCR with EvaGreen assay. (A) Schematic of the region of interest (ROI, upper) for copy number variant analysis and the reference (REF, lower). (B) Schematic of the ddPCR with EvaGreen workflow.

Article Snippet: DNA Nuclease free water 2x ddPCR EvaGreen Supermix (includes hot start DNA polymerase, dNTPs including dUTP; Bio-Rad) 20x ROI target primer (see ) 20x REF target (RPP30) primer/Taqman probe mix (see Reagents and Solutions recipe) 2x buffer control for EvaGreen (Bio-Rad) Droplet Generation Oil for Evagreen (Bio-Rad) 96-well plates 96-well plate centrifuge DG8 droplet generator cartridges (single-use; Bio-Rad) DG8 droplet generator cartridge holder (Bio-Rad) DdPCR droplet generation (DG) oil (Bio-Rad) DG8 gaskets (single-use; Bio-Rad) QX200 droplet generator (Bio-Rad) Eppendorf twin.tec semi-skirted 96 well plate Heat sealer Heat sealing PCR foil Thermal cycler Bio-Rad QX200 droplet reader QuantaSoft software

Techniques: Variant Assay

Journal: Current protocols in human genetics

Article Title: Droplet Digital PCR with EvaGreen Assay: Confirmational Analysis of Structural Variants

doi: 10.1002/cphg.58

Figure Lengend Snippet: Sample results from ddPCR with EvaGreen assay. (A) 1D plots for FAM (upper) and VIC (lower) channels. (B) 2D plot showing combined three clusters of droplets: blue, EvaGreen-positive (ROI); orange, EvaGreen- and VIC-positive(i.e., REF gene RRP30); and black, negative.

Article Snippet: DNA Nuclease free water 2x ddPCR EvaGreen Supermix (includes hot start DNA polymerase, dNTPs including dUTP; Bio-Rad) 20x ROI target primer (see ) 20x REF target (RPP30) primer/Taqman probe mix (see Reagents and Solutions recipe) 2x buffer control for EvaGreen (Bio-Rad) Droplet Generation Oil for Evagreen (Bio-Rad) 96-well plates 96-well plate centrifuge DG8 droplet generator cartridges (single-use; Bio-Rad) DG8 droplet generator cartridge holder (Bio-Rad) DdPCR droplet generation (DG) oil (Bio-Rad) DG8 gaskets (single-use; Bio-Rad) QX200 droplet generator (Bio-Rad) Eppendorf twin.tec semi-skirted 96 well plate Heat sealer Heat sealing PCR foil Thermal cycler Bio-Rad QX200 droplet reader QuantaSoft software

Techniques:

Journal: Current protocols in human genetics

Article Title: Droplet Digital PCR with EvaGreen Assay: Confirmational Analysis of Structural Variants

doi: 10.1002/cphg.58

Figure Lengend Snippet: Sample CNV Results using ddPCR with EvaGreen Assa

Article Snippet: DNA Nuclease free water 2x ddPCR EvaGreen Supermix (includes hot start DNA polymerase, dNTPs including dUTP; Bio-Rad) 20x ROI target primer (see ) 20x REF target (RPP30) primer/Taqman probe mix (see Reagents and Solutions recipe) 2x buffer control for EvaGreen (Bio-Rad) Droplet Generation Oil for Evagreen (Bio-Rad) 96-well plates 96-well plate centrifuge DG8 droplet generator cartridges (single-use; Bio-Rad) DG8 droplet generator cartridge holder (Bio-Rad) DdPCR droplet generation (DG) oil (Bio-Rad) DG8 gaskets (single-use; Bio-Rad) QX200 droplet generator (Bio-Rad) Eppendorf twin.tec semi-skirted 96 well plate Heat sealer Heat sealing PCR foil Thermal cycler Bio-Rad QX200 droplet reader QuantaSoft software

Techniques: