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Tissue <t>DNA</t> inhibition for SIV viral quantification. DNA was extracted from 7 tissues from a necropsied Rhesus macaque (310–08) that was infected with SIVmac239 (see Methods section for details). DNA samples (undiluted or 1:10 diluted) were first subject to preamplification, and 10% of each preamp product was subject to duplex <t>qPCR</t> of Rhesus macaque SIV gag and CCR5 [ 14 ]. 1000 copies of SIV DNA standard was spiked into one reaction containing the preamp product to assess PCR inhibition in tissue DNA, while a buffer spike (1000 copies of SIV DNA standard spiked into buffer) served as “no inhibition” control. SIV gag spike copy (lower left) for each sample was derived from the SIV gag spike Ct (upper left; compared to buffer spike Ct), while CCR5 average copies (single stranded) were derived from a CCR5 standard curve (upper right). In addition, the CCR5 copies (single stranded) were independently determined from the DNA input into each reaction (lower right) based on DNA concentration measurement using NanoDrop.
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1) Product Images from "Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays"

Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

Journal: PLoS ONE

doi: 10.1371/journal.pone.0233085

Tissue DNA inhibition for SIV viral quantification. DNA was extracted from 7 tissues from a necropsied Rhesus macaque (310–08) that was infected with SIVmac239 (see Methods section for details). DNA samples (undiluted or 1:10 diluted) were first subject to preamplification, and 10% of each preamp product was subject to duplex qPCR of Rhesus macaque SIV gag and CCR5 [ 14 ]. 1000 copies of SIV DNA standard was spiked into one reaction containing the preamp product to assess PCR inhibition in tissue DNA, while a buffer spike (1000 copies of SIV DNA standard spiked into buffer) served as “no inhibition” control. SIV gag spike copy (lower left) for each sample was derived from the SIV gag spike Ct (upper left; compared to buffer spike Ct), while CCR5 average copies (single stranded) were derived from a CCR5 standard curve (upper right). In addition, the CCR5 copies (single stranded) were independently determined from the DNA input into each reaction (lower right) based on DNA concentration measurement using NanoDrop.
Figure Legend Snippet: Tissue DNA inhibition for SIV viral quantification. DNA was extracted from 7 tissues from a necropsied Rhesus macaque (310–08) that was infected with SIVmac239 (see Methods section for details). DNA samples (undiluted or 1:10 diluted) were first subject to preamplification, and 10% of each preamp product was subject to duplex qPCR of Rhesus macaque SIV gag and CCR5 [ 14 ]. 1000 copies of SIV DNA standard was spiked into one reaction containing the preamp product to assess PCR inhibition in tissue DNA, while a buffer spike (1000 copies of SIV DNA standard spiked into buffer) served as “no inhibition” control. SIV gag spike copy (lower left) for each sample was derived from the SIV gag spike Ct (upper left; compared to buffer spike Ct), while CCR5 average copies (single stranded) were derived from a CCR5 standard curve (upper right). In addition, the CCR5 copies (single stranded) were independently determined from the DNA input into each reaction (lower right) based on DNA concentration measurement using NanoDrop.

Techniques Used: Inhibition, Infection, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Derivative Assay, Concentration Assay

ddPCR optimization based on existing SIV gag DNA real time qPCR assay and condition. (A) Direct migration of the qPCR SIV gag DNA assay in existing format onto RainDance ddPCR platform. Left: tissue DNA containing preamplified SIV DNA as template; Right: SIV DNA standard spike-in in buffer as template. (B) The effect of modifying MgCl 2 concentration on cluster separation. (C) Background issue in the SIV target detection region using existing assay’s master mix with optimized MgCl 2 concentration.
Figure Legend Snippet: ddPCR optimization based on existing SIV gag DNA real time qPCR assay and condition. (A) Direct migration of the qPCR SIV gag DNA assay in existing format onto RainDance ddPCR platform. Left: tissue DNA containing preamplified SIV DNA as template; Right: SIV DNA standard spike-in in buffer as template. (B) The effect of modifying MgCl 2 concentration on cluster separation. (C) Background issue in the SIV target detection region using existing assay’s master mix with optimized MgCl 2 concentration.

Techniques Used: Real-time Polymerase Chain Reaction, Migration, Concentration Assay

Comparison between ddPCR and qPCR quantitation results in low viral DNA range
Figure Legend Snippet: Comparison between ddPCR and qPCR quantitation results in low viral DNA range

Techniques Used: Real-time Polymerase Chain Reaction, Quantitation Assay

SIV ddPCR DNA assay characterization. (A) Sample DNA input tolerance at the droplet formation step. Droplet integrity was monitored by examining a portion of the droplets in each lane as they moved through the Source instrument during dropletization. In addition, total droplet number for each input level after dropletization (retrieved from the “RainDrop Run Completion” screen) served as another indicator of sample DNA input tolerance. DNA sample used was duodenum DNA from Rhesus macaque 313–08. (B) Total number of droplets generated during dropletization remains constant in the range of tissue DNA amount tested (1 million to 8 million cell equivalent input). Total droplet number for each sample after dropletization serves as an additional indicator of sample DNA input tolerance. Note that for each level of DNA input, only a fraction (~1.6%) of the droplets were counted for QC purpose by the Source machine. (C) Estimation of the limit of detection (LoD) of the ddPCR assay based on the Digital MIQE Guidelines [ 7 ]. According to the guidelines, when running costs preclude optimization using ddPCR, qPCR can be used to determine certain assay parameters. (D) Performance of the SIV ddPCR assay in TaqMan genotyping mastermix in qPCR format. SIV gag DNA standard was diluted with buffer diluent. The standards were assayed as described in Materials and Methods in the following replicate format: 1 million down to 100 copies input per reaction: each in triplicates; 50, 20, 10, 7 and 5 copies input per reaction: each in 10 replicates. The data were plotted and analyzed according to the routine analyses provided in the software package with the ABI 7500 SDS instrument. (E) Instead of measuring 60 ddPCR replicates to obtain 95% confidence, we obtained an approximate estimation of the LoD using a lower number of ddPCR reaction replicates and required the false negative rate to be below 5% (i.e. all 10 replicates have to be positive).
Figure Legend Snippet: SIV ddPCR DNA assay characterization. (A) Sample DNA input tolerance at the droplet formation step. Droplet integrity was monitored by examining a portion of the droplets in each lane as they moved through the Source instrument during dropletization. In addition, total droplet number for each input level after dropletization (retrieved from the “RainDrop Run Completion” screen) served as another indicator of sample DNA input tolerance. DNA sample used was duodenum DNA from Rhesus macaque 313–08. (B) Total number of droplets generated during dropletization remains constant in the range of tissue DNA amount tested (1 million to 8 million cell equivalent input). Total droplet number for each sample after dropletization serves as an additional indicator of sample DNA input tolerance. Note that for each level of DNA input, only a fraction (~1.6%) of the droplets were counted for QC purpose by the Source machine. (C) Estimation of the limit of detection (LoD) of the ddPCR assay based on the Digital MIQE Guidelines [ 7 ]. According to the guidelines, when running costs preclude optimization using ddPCR, qPCR can be used to determine certain assay parameters. (D) Performance of the SIV ddPCR assay in TaqMan genotyping mastermix in qPCR format. SIV gag DNA standard was diluted with buffer diluent. The standards were assayed as described in Materials and Methods in the following replicate format: 1 million down to 100 copies input per reaction: each in triplicates; 50, 20, 10, 7 and 5 copies input per reaction: each in 10 replicates. The data were plotted and analyzed according to the routine analyses provided in the software package with the ABI 7500 SDS instrument. (E) Instead of measuring 60 ddPCR replicates to obtain 95% confidence, we obtained an approximate estimation of the LoD using a lower number of ddPCR reaction replicates and required the false negative rate to be below 5% (i.e. all 10 replicates have to be positive).

Techniques Used: Generated, Real-time Polymerase Chain Reaction, Software

ddPCR and qPCR comparison. (A) Sample inhibition comparison between ddPCR and qPCR. An ovarian DNA sample (Rhesus macaque 311–08) was subjected to nested (i.e. preamplified) qPCR analysis or direct RainDance (“RD” in the table header) ddPCR analysis for SIV DNA viral load. At 3.7 million cell input per reaction (at the preamp step), SIV qPCR signal is inhibited by 99%, while at 4 M cell input per direct ddPCR reaction, SIV signal is not inhibited. Note that in the qPCR graph, the data point (32.6 ± 5.4) on the left (0.37 million cell input per reaction) was derived from a standard curve, while the data point (4.0) on the right (3.7 million cell input per reaction) was derived from Poisson statistics (i.e. DNA copies derived from the positive well rate among the replicate reactions). (B) Quantification of Rhesus macaque necropsy tissue DNA samples (from Rhesus macaque 27882) (including an uninfected negative control sample) for SIV DNA using ddPCR. (C) Comparison between ddPCR and nested qPCR analysis results for the necropsy tissue samples in (B). RD, RainDance. S.D., standard deviation.
Figure Legend Snippet: ddPCR and qPCR comparison. (A) Sample inhibition comparison between ddPCR and qPCR. An ovarian DNA sample (Rhesus macaque 311–08) was subjected to nested (i.e. preamplified) qPCR analysis or direct RainDance (“RD” in the table header) ddPCR analysis for SIV DNA viral load. At 3.7 million cell input per reaction (at the preamp step), SIV qPCR signal is inhibited by 99%, while at 4 M cell input per direct ddPCR reaction, SIV signal is not inhibited. Note that in the qPCR graph, the data point (32.6 ± 5.4) on the left (0.37 million cell input per reaction) was derived from a standard curve, while the data point (4.0) on the right (3.7 million cell input per reaction) was derived from Poisson statistics (i.e. DNA copies derived from the positive well rate among the replicate reactions). (B) Quantification of Rhesus macaque necropsy tissue DNA samples (from Rhesus macaque 27882) (including an uninfected negative control sample) for SIV DNA using ddPCR. (C) Comparison between ddPCR and nested qPCR analysis results for the necropsy tissue samples in (B). RD, RainDance. S.D., standard deviation.

Techniques Used: Real-time Polymerase Chain Reaction, Inhibition, Derivative Assay, Negative Control, Standard Deviation

Identifying an optimal ddPCR assay format and condition. Performance of an MGB probe-based SIV gag ddPCR assay using TaqMan genotyping mastermix on (A) SIV (on CCR5 background) spike-in templates and (B) ovary tissue DNA from a Rhesus macaque (311–08) infected with SIVmac239. (C) A summary of all other tested conditions (probe and mastermix) and the observed issue(s) associated with each condition.
Figure Legend Snippet: Identifying an optimal ddPCR assay format and condition. Performance of an MGB probe-based SIV gag ddPCR assay using TaqMan genotyping mastermix on (A) SIV (on CCR5 background) spike-in templates and (B) ovary tissue DNA from a Rhesus macaque (311–08) infected with SIVmac239. (C) A summary of all other tested conditions (probe and mastermix) and the observed issue(s) associated with each condition.

Techniques Used: Infection

SIV ddPCR DNA assay performance. (A) SIV ddPCR DNA assay detection of low single digit level SIV DNA input and linear dynamic range of the SIV ddPCR DNA assay. Different amount of SIV DNA template was spiked in 1 million Rhesus macaque PBMC equivalent of genomic DNA background each. SIV DNA amount was quantified by direct ddPCR. (B) Quantification data of Fig 5A.
Figure Legend Snippet: SIV ddPCR DNA assay performance. (A) SIV ddPCR DNA assay detection of low single digit level SIV DNA input and linear dynamic range of the SIV ddPCR DNA assay. Different amount of SIV DNA template was spiked in 1 million Rhesus macaque PBMC equivalent of genomic DNA background each. SIV DNA amount was quantified by direct ddPCR. (B) Quantification data of Fig 5A.

Techniques Used:

2) Product Images from "Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays"

Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

Journal: PLoS ONE

doi: 10.1371/journal.pone.0233085

ddPCR optimization based on existing SIV gag DNA real time qPCR assay and condition. (A) Direct migration of the qPCR SIV gag DNA assay in existing format onto RainDance ddPCR platform. Left: tissue DNA containing preamplified SIV DNA as template; Right: SIV DNA standard spike-in in buffer as template. (B) The effect of modifying MgCl 2 concentration on cluster separation. (C) Background issue in the SIV target detection region using existing assay’s master mix with optimized MgCl 2 concentration.
Figure Legend Snippet: ddPCR optimization based on existing SIV gag DNA real time qPCR assay and condition. (A) Direct migration of the qPCR SIV gag DNA assay in existing format onto RainDance ddPCR platform. Left: tissue DNA containing preamplified SIV DNA as template; Right: SIV DNA standard spike-in in buffer as template. (B) The effect of modifying MgCl 2 concentration on cluster separation. (C) Background issue in the SIV target detection region using existing assay’s master mix with optimized MgCl 2 concentration.

Techniques Used: Real-time Polymerase Chain Reaction, Migration, Concentration Assay

ddPCR and qPCR comparison. (A) Sample inhibition comparison between ddPCR and qPCR. An ovarian DNA sample (Rhesus macaque 311–08) was subjected to nested (i.e. preamplified) qPCR analysis or direct RainDance (“RD” in the table header) ddPCR analysis for SIV DNA viral load. At 3.7 million cell input per reaction (at the preamp step), SIV qPCR signal is inhibited by 99%, while at 4 M cell input per direct ddPCR reaction, SIV signal is not inhibited. Note that in the qPCR graph, the data point (32.6 ± 5.4) on the left (0.37 million cell input per reaction) was derived from a standard curve, while the data point (4.0) on the right (3.7 million cell input per reaction) was derived from Poisson statistics (i.e. DNA copies derived from the positive well rate among the replicate reactions). (B) Quantification of Rhesus macaque necropsy tissue DNA samples (from Rhesus macaque 27882) (including an uninfected negative control sample) for SIV DNA using ddPCR. (C) Comparison between ddPCR and nested qPCR analysis results for the necropsy tissue samples in (B). RD, RainDance. S.D., standard deviation.
Figure Legend Snippet: ddPCR and qPCR comparison. (A) Sample inhibition comparison between ddPCR and qPCR. An ovarian DNA sample (Rhesus macaque 311–08) was subjected to nested (i.e. preamplified) qPCR analysis or direct RainDance (“RD” in the table header) ddPCR analysis for SIV DNA viral load. At 3.7 million cell input per reaction (at the preamp step), SIV qPCR signal is inhibited by 99%, while at 4 M cell input per direct ddPCR reaction, SIV signal is not inhibited. Note that in the qPCR graph, the data point (32.6 ± 5.4) on the left (0.37 million cell input per reaction) was derived from a standard curve, while the data point (4.0) on the right (3.7 million cell input per reaction) was derived from Poisson statistics (i.e. DNA copies derived from the positive well rate among the replicate reactions). (B) Quantification of Rhesus macaque necropsy tissue DNA samples (from Rhesus macaque 27882) (including an uninfected negative control sample) for SIV DNA using ddPCR. (C) Comparison between ddPCR and nested qPCR analysis results for the necropsy tissue samples in (B). RD, RainDance. S.D., standard deviation.

Techniques Used: Real-time Polymerase Chain Reaction, Inhibition, Derivative Assay, Negative Control, Standard Deviation

3) Product Images from "Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays"

Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

Journal: PLoS ONE

doi: 10.1371/journal.pone.0233085

ddPCR optimization based on existing SIV gag DNA real time qPCR assay and condition. (A) Direct migration of the qPCR SIV gag DNA assay in existing format onto RainDance ddPCR platform. Left: tissue DNA containing preamplified SIV DNA as template; Right: SIV DNA standard spike-in in buffer as template. (B) The effect of modifying MgCl 2 concentration on cluster separation. (C) Background issue in the SIV target detection region using existing assay’s master mix with optimized MgCl 2 concentration.
Figure Legend Snippet: ddPCR optimization based on existing SIV gag DNA real time qPCR assay and condition. (A) Direct migration of the qPCR SIV gag DNA assay in existing format onto RainDance ddPCR platform. Left: tissue DNA containing preamplified SIV DNA as template; Right: SIV DNA standard spike-in in buffer as template. (B) The effect of modifying MgCl 2 concentration on cluster separation. (C) Background issue in the SIV target detection region using existing assay’s master mix with optimized MgCl 2 concentration.

Techniques Used: Real-time Polymerase Chain Reaction, Migration, Concentration Assay

Comparison between ddPCR and qPCR quantitation results in low viral DNA range
Figure Legend Snippet: Comparison between ddPCR and qPCR quantitation results in low viral DNA range

Techniques Used: Real-time Polymerase Chain Reaction, Quantitation Assay

SIV ddPCR DNA assay characterization. (A) Sample DNA input tolerance at the droplet formation step. Droplet integrity was monitored by examining a portion of the droplets in each lane as they moved through the Source instrument during dropletization. In addition, total droplet number for each input level after dropletization (retrieved from the “RainDrop Run Completion” screen) served as another indicator of sample DNA input tolerance. DNA sample used was duodenum DNA from Rhesus macaque 313–08. (B) Total number of droplets generated during dropletization remains constant in the range of tissue DNA amount tested (1 million to 8 million cell equivalent input). Total droplet number for each sample after dropletization serves as an additional indicator of sample DNA input tolerance. Note that for each level of DNA input, only a fraction (~1.6%) of the droplets were counted for QC purpose by the Source machine. (C) Estimation of the limit of detection (LoD) of the ddPCR assay based on the Digital MIQE Guidelines [ 7 ]. According to the guidelines, when running costs preclude optimization using ddPCR, qPCR can be used to determine certain assay parameters. (D) Performance of the SIV ddPCR assay in TaqMan genotyping mastermix in qPCR format. SIV gag DNA standard was diluted with buffer diluent. The standards were assayed as described in Materials and Methods in the following replicate format: 1 million down to 100 copies input per reaction: each in triplicates; 50, 20, 10, 7 and 5 copies input per reaction: each in 10 replicates. The data were plotted and analyzed according to the routine analyses provided in the software package with the ABI 7500 SDS instrument. (E) Instead of measuring 60 ddPCR replicates to obtain 95% confidence, we obtained an approximate estimation of the LoD using a lower number of ddPCR reaction replicates and required the false negative rate to be below 5% (i.e. all 10 replicates have to be positive).
Figure Legend Snippet: SIV ddPCR DNA assay characterization. (A) Sample DNA input tolerance at the droplet formation step. Droplet integrity was monitored by examining a portion of the droplets in each lane as they moved through the Source instrument during dropletization. In addition, total droplet number for each input level after dropletization (retrieved from the “RainDrop Run Completion” screen) served as another indicator of sample DNA input tolerance. DNA sample used was duodenum DNA from Rhesus macaque 313–08. (B) Total number of droplets generated during dropletization remains constant in the range of tissue DNA amount tested (1 million to 8 million cell equivalent input). Total droplet number for each sample after dropletization serves as an additional indicator of sample DNA input tolerance. Note that for each level of DNA input, only a fraction (~1.6%) of the droplets were counted for QC purpose by the Source machine. (C) Estimation of the limit of detection (LoD) of the ddPCR assay based on the Digital MIQE Guidelines [ 7 ]. According to the guidelines, when running costs preclude optimization using ddPCR, qPCR can be used to determine certain assay parameters. (D) Performance of the SIV ddPCR assay in TaqMan genotyping mastermix in qPCR format. SIV gag DNA standard was diluted with buffer diluent. The standards were assayed as described in Materials and Methods in the following replicate format: 1 million down to 100 copies input per reaction: each in triplicates; 50, 20, 10, 7 and 5 copies input per reaction: each in 10 replicates. The data were plotted and analyzed according to the routine analyses provided in the software package with the ABI 7500 SDS instrument. (E) Instead of measuring 60 ddPCR replicates to obtain 95% confidence, we obtained an approximate estimation of the LoD using a lower number of ddPCR reaction replicates and required the false negative rate to be below 5% (i.e. all 10 replicates have to be positive).

Techniques Used: Generated, Real-time Polymerase Chain Reaction, Software

ddPCR and qPCR comparison. (A) Sample inhibition comparison between ddPCR and qPCR. An ovarian DNA sample (Rhesus macaque 311–08) was subjected to nested (i.e. preamplified) qPCR analysis or direct RainDance (“RD” in the table header) ddPCR analysis for SIV DNA viral load. At 3.7 million cell input per reaction (at the preamp step), SIV qPCR signal is inhibited by 99%, while at 4 M cell input per direct ddPCR reaction, SIV signal is not inhibited. Note that in the qPCR graph, the data point (32.6 ± 5.4) on the left (0.37 million cell input per reaction) was derived from a standard curve, while the data point (4.0) on the right (3.7 million cell input per reaction) was derived from Poisson statistics (i.e. DNA copies derived from the positive well rate among the replicate reactions). (B) Quantification of Rhesus macaque necropsy tissue DNA samples (from Rhesus macaque 27882) (including an uninfected negative control sample) for SIV DNA using ddPCR. (C) Comparison between ddPCR and nested qPCR analysis results for the necropsy tissue samples in (B). RD, RainDance. S.D., standard deviation.
Figure Legend Snippet: ddPCR and qPCR comparison. (A) Sample inhibition comparison between ddPCR and qPCR. An ovarian DNA sample (Rhesus macaque 311–08) was subjected to nested (i.e. preamplified) qPCR analysis or direct RainDance (“RD” in the table header) ddPCR analysis for SIV DNA viral load. At 3.7 million cell input per reaction (at the preamp step), SIV qPCR signal is inhibited by 99%, while at 4 M cell input per direct ddPCR reaction, SIV signal is not inhibited. Note that in the qPCR graph, the data point (32.6 ± 5.4) on the left (0.37 million cell input per reaction) was derived from a standard curve, while the data point (4.0) on the right (3.7 million cell input per reaction) was derived from Poisson statistics (i.e. DNA copies derived from the positive well rate among the replicate reactions). (B) Quantification of Rhesus macaque necropsy tissue DNA samples (from Rhesus macaque 27882) (including an uninfected negative control sample) for SIV DNA using ddPCR. (C) Comparison between ddPCR and nested qPCR analysis results for the necropsy tissue samples in (B). RD, RainDance. S.D., standard deviation.

Techniques Used: Real-time Polymerase Chain Reaction, Inhibition, Derivative Assay, Negative Control, Standard Deviation

Identifying an optimal ddPCR assay format and condition. Performance of an MGB probe-based SIV gag ddPCR assay using TaqMan genotyping mastermix on (A) SIV (on CCR5 background) spike-in templates and (B) ovary tissue DNA from a Rhesus macaque (311–08) infected with SIVmac239. (C) A summary of all other tested conditions (probe and mastermix) and the observed issue(s) associated with each condition.
Figure Legend Snippet: Identifying an optimal ddPCR assay format and condition. Performance of an MGB probe-based SIV gag ddPCR assay using TaqMan genotyping mastermix on (A) SIV (on CCR5 background) spike-in templates and (B) ovary tissue DNA from a Rhesus macaque (311–08) infected with SIVmac239. (C) A summary of all other tested conditions (probe and mastermix) and the observed issue(s) associated with each condition.

Techniques Used: Infection

SIV ddPCR DNA assay performance. (A) SIV ddPCR DNA assay detection of low single digit level SIV DNA input and linear dynamic range of the SIV ddPCR DNA assay. Different amount of SIV DNA template was spiked in 1 million Rhesus macaque PBMC equivalent of genomic DNA background each. SIV DNA amount was quantified by direct ddPCR. (B) Quantification data of Fig 5A.
Figure Legend Snippet: SIV ddPCR DNA assay performance. (A) SIV ddPCR DNA assay detection of low single digit level SIV DNA input and linear dynamic range of the SIV ddPCR DNA assay. Different amount of SIV DNA template was spiked in 1 million Rhesus macaque PBMC equivalent of genomic DNA background each. SIV DNA amount was quantified by direct ddPCR. (B) Quantification data of Fig 5A.

Techniques Used:

4) Product Images from "Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6"

Article Title: Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6

Journal: eLife

doi: 10.7554/eLife.27873

Measuring translocations after HBB and AAVS1 di-genic targeting. ( a ) Schematic showing the HBB gene on chromosome 11 and the AAVS1 locus on chromosome 19. The Cas9 cut sites are shown in red. One of the two possible monocentric translocations is shown. ( b ) The reference sequence of the HBB-AAVS1 translocation is shown in the top. Below are representative translocation sequences from GFP - BFP - HSPCs sorted seven days after targeting (see Figure 3e , left panel). ( c ) Representative ddPCR analyses quantifying translocations in NTC (non-template control), mock-electroporated, and GFP - BFP - cells (see Figure 3e , right panel). The reference assay quantifies TERT gene copies used to normalize for DNA input. The translocation assay probe binds 50 bp away from the junction and none of the identified translocations would therefore exclude probe binding.
Figure Legend Snippet: Measuring translocations after HBB and AAVS1 di-genic targeting. ( a ) Schematic showing the HBB gene on chromosome 11 and the AAVS1 locus on chromosome 19. The Cas9 cut sites are shown in red. One of the two possible monocentric translocations is shown. ( b ) The reference sequence of the HBB-AAVS1 translocation is shown in the top. Below are representative translocation sequences from GFP - BFP - HSPCs sorted seven days after targeting (see Figure 3e , left panel). ( c ) Representative ddPCR analyses quantifying translocations in NTC (non-template control), mock-electroporated, and GFP - BFP - cells (see Figure 3e , right panel). The reference assay quantifies TERT gene copies used to normalize for DNA input. The translocation assay probe binds 50 bp away from the junction and none of the identified translocations would therefore exclude probe binding.

Techniques Used: Sequencing, Translocation Assay, Binding Assay

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Real-time Polymerase Chain Reaction:

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Article Snippet: RNA was DNase treated (Invitrogen) prior to cDNA synthesis. .. 100 ng of RNA was used as template in each quantitative real-time PCR (qRT-PCR) reaction. cDNA synthesis control was performed to ensure the absence of DNA contamination. qRT-PCR was carried out using iTaq™ Universal SYBR® Green Supermix (Bio-Rad) and Bio-Rad CFX96 qPCR detection system. .. Primers for test and control transcripts were designed using boundary sequence of two exons to avoid any amplification from genomic DNA contamination (Additional file : Table S1). qRT-PCR was done in biological triplicates with actin and tubulin as the endogenous control.

Quantitative RT-PCR:

Article Title: Disruption of zinc finger DNA binding domain in catabolite repressor Mig1 increases growth rate, hyphal branching, and cellulase expression in hypercellulolytic fungus Penicillium funiculosum NCIM1228
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SYBR Green Assay:

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Article Snippet: Quantification of HBV RNA and DNA HBV RNA from the transfected cell supernatant was detected by TaqMan qPCR following the protocols previously described ( ). .. Total supernatant DNA, cellular RNA, and DNA were detected using SYBR Green fluorescence (BioRad, Cat# 1725124). .. An HBV-containing plasmid was used as a positive control and to produce the dilution series for the qPCR standards; water and “mock extracted” template and mock transfection were used as negative controls.

AST Assay:

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Article Snippet: Although the minimum incubation time is mainly dependent on the pathogen–drug combination, it can be reduced by using digital methods with high-quantification resolution capable of detecting a smaller change in bacterial nucleic acids. .. The scheme of digital AST was initially demonstrated by Schoepp et al. ( ) who performed digital quantification of DNA replication using droplet digital PCR (the QX200 system by Bio-Rad). .. To illustrate this capability, a recent method proposed by Athamanolap et al. ( ) implemented 16S PCR and HRMA to perform integrated bacterial identification and antibiotic susceptibility testing ( ).

Digital PCR:

Article Title: Emerging Analytical Techniques for Rapid Pathogen Identification and Susceptibility Testing
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Electrophoresis:

Article Title: Opine-Based Agrobacterium Competitiveness: Dual Expression Control of the Agrocinopine Catabolism (acc) Operon by Agrocinopines and Phosphate Levels
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Fluorescence:

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Infection:

Article Title: Diverse Roles for E4orf3 at Late Times of Infection Revealed in an E1B 55-Kilodalton Protein Mutant Background
Article Snippet: The amount of bound radioactive DNA was quantified by phosphorescence imaging (ImageQuant software; Molecular Dynamics, Sunnyvale, Calif.), and the amount of adenovirus DNA per cell was expressed relative to the amount measured in infected HeLa cells. .. To detect the formation of viral DNA concatemers, total DNA from infected cells was analyzed by pulsed-field gel electrophoresis with a Bio-Rad CHEF DR II instrument (Bio-Rad, Hercules, Calif.) as described elsewhere ( , ). .. In brief, 106 infected cells were detached with trypsin 24 or 48 h after infection and suspended in 0.25 ml of PBS, which was immediately mixed with 0.25 ml of liquid 1.5% low-melting-point agarose in PBS.

Pulsed-Field Gel:

Article Title: Diverse Roles for E4orf3 at Late Times of Infection Revealed in an E1B 55-Kilodalton Protein Mutant Background
Article Snippet: The amount of bound radioactive DNA was quantified by phosphorescence imaging (ImageQuant software; Molecular Dynamics, Sunnyvale, Calif.), and the amount of adenovirus DNA per cell was expressed relative to the amount measured in infected HeLa cells. .. To detect the formation of viral DNA concatemers, total DNA from infected cells was analyzed by pulsed-field gel electrophoresis with a Bio-Rad CHEF DR II instrument (Bio-Rad, Hercules, Calif.) as described elsewhere ( , ). .. In brief, 106 infected cells were detached with trypsin 24 or 48 h after infection and suspended in 0.25 ml of PBS, which was immediately mixed with 0.25 ml of liquid 1.5% low-melting-point agarose in PBS.

Polymerase Chain Reaction:

Article Title: Genomic Characterization of the Filamentous Integrative Bacteriophages ?RSS1 and ?RSM1, Which Infect Ralstonia solanacearum ▿
Article Snippet: Approximately 2,160 bases of φRSS1 DNA lacking the modules for structural proteins and morphogenesis (ORF4 to ORF11) was amplified by PCR with a forward primer, 5′-CGG AAT TCT ATC CGG AGT AAC GA AAA G, corresponding to the 3′ end of orf11 and a reverse primer, 5′-ATG GAA TTC TCC TTG AGA TGG AGG TTG AG, corresponding to the 5′ end of orf4 . .. Twenty-five rounds of PCR were performed with RF DNA (1 ng) of φRSS1 as a template under the standard conditions in a MY Cycler (Bio-Rad). .. After digestion with EcoRI at the primer sites, the amplified fragment was connected to a Kmr cassette cut out with EcoRI from plasmid pUC4-KIXX (Amersham Biosciences).

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    Bio-Rad input dna
    ddPCR optimization based on existing SIV gag <t>DNA</t> real time qPCR assay and condition. (A) Direct migration of the qPCR SIV gag DNA assay in existing format onto <t>RainDance</t> ddPCR platform. Left: tissue DNA containing preamplified SIV DNA as template; Right: SIV DNA standard spike-in in buffer as template. (B) The effect of modifying MgCl 2 concentration on cluster separation. (C) Background issue in the SIV target detection region using existing assay’s master mix with optimized MgCl 2 concentration.
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    ddPCR optimization based on existing SIV gag DNA real time qPCR assay and condition. (A) Direct migration of the qPCR SIV gag DNA assay in existing format onto RainDance ddPCR platform. Left: tissue DNA containing preamplified SIV DNA as template; Right: SIV DNA standard spike-in in buffer as template. (B) The effect of modifying MgCl 2 concentration on cluster separation. (C) Background issue in the SIV target detection region using existing assay’s master mix with optimized MgCl 2 concentration.

    Journal: PLoS ONE

    Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

    doi: 10.1371/journal.pone.0233085

    Figure Lengend Snippet: ddPCR optimization based on existing SIV gag DNA real time qPCR assay and condition. (A) Direct migration of the qPCR SIV gag DNA assay in existing format onto RainDance ddPCR platform. Left: tissue DNA containing preamplified SIV DNA as template; Right: SIV DNA standard spike-in in buffer as template. (B) The effect of modifying MgCl 2 concentration on cluster separation. (C) Background issue in the SIV target detection region using existing assay’s master mix with optimized MgCl 2 concentration.

    Article Snippet: In conclusion, combining two ddPCR assays for SIV nucleic acids detection with the RainDance ddPCR platform can enable a large amount of input DNA to be analyzed per reaction, and can overcome severe RNA inhibition when combined with suitable reverse transcription enzyme(s).

    Techniques: Real-time Polymerase Chain Reaction, Migration, Concentration Assay

    Comparison between ddPCR and qPCR quantitation results in low viral DNA range

    Journal: PLoS ONE

    Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

    doi: 10.1371/journal.pone.0233085

    Figure Lengend Snippet: Comparison between ddPCR and qPCR quantitation results in low viral DNA range

    Article Snippet: In conclusion, combining two ddPCR assays for SIV nucleic acids detection with the RainDance ddPCR platform can enable a large amount of input DNA to be analyzed per reaction, and can overcome severe RNA inhibition when combined with suitable reverse transcription enzyme(s).

    Techniques: Real-time Polymerase Chain Reaction, Quantitation Assay

    SIV ddPCR DNA assay characterization. (A) Sample DNA input tolerance at the droplet formation step. Droplet integrity was monitored by examining a portion of the droplets in each lane as they moved through the Source instrument during dropletization. In addition, total droplet number for each input level after dropletization (retrieved from the “RainDrop Run Completion” screen) served as another indicator of sample DNA input tolerance. DNA sample used was duodenum DNA from Rhesus macaque 313–08. (B) Total number of droplets generated during dropletization remains constant in the range of tissue DNA amount tested (1 million to 8 million cell equivalent input). Total droplet number for each sample after dropletization serves as an additional indicator of sample DNA input tolerance. Note that for each level of DNA input, only a fraction (~1.6%) of the droplets were counted for QC purpose by the Source machine. (C) Estimation of the limit of detection (LoD) of the ddPCR assay based on the Digital MIQE Guidelines [ 7 ]. According to the guidelines, when running costs preclude optimization using ddPCR, qPCR can be used to determine certain assay parameters. (D) Performance of the SIV ddPCR assay in TaqMan genotyping mastermix in qPCR format. SIV gag DNA standard was diluted with buffer diluent. The standards were assayed as described in Materials and Methods in the following replicate format: 1 million down to 100 copies input per reaction: each in triplicates; 50, 20, 10, 7 and 5 copies input per reaction: each in 10 replicates. The data were plotted and analyzed according to the routine analyses provided in the software package with the ABI 7500 SDS instrument. (E) Instead of measuring 60 ddPCR replicates to obtain 95% confidence, we obtained an approximate estimation of the LoD using a lower number of ddPCR reaction replicates and required the false negative rate to be below 5% (i.e. all 10 replicates have to be positive).

    Journal: PLoS ONE

    Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

    doi: 10.1371/journal.pone.0233085

    Figure Lengend Snippet: SIV ddPCR DNA assay characterization. (A) Sample DNA input tolerance at the droplet formation step. Droplet integrity was monitored by examining a portion of the droplets in each lane as they moved through the Source instrument during dropletization. In addition, total droplet number for each input level after dropletization (retrieved from the “RainDrop Run Completion” screen) served as another indicator of sample DNA input tolerance. DNA sample used was duodenum DNA from Rhesus macaque 313–08. (B) Total number of droplets generated during dropletization remains constant in the range of tissue DNA amount tested (1 million to 8 million cell equivalent input). Total droplet number for each sample after dropletization serves as an additional indicator of sample DNA input tolerance. Note that for each level of DNA input, only a fraction (~1.6%) of the droplets were counted for QC purpose by the Source machine. (C) Estimation of the limit of detection (LoD) of the ddPCR assay based on the Digital MIQE Guidelines [ 7 ]. According to the guidelines, when running costs preclude optimization using ddPCR, qPCR can be used to determine certain assay parameters. (D) Performance of the SIV ddPCR assay in TaqMan genotyping mastermix in qPCR format. SIV gag DNA standard was diluted with buffer diluent. The standards were assayed as described in Materials and Methods in the following replicate format: 1 million down to 100 copies input per reaction: each in triplicates; 50, 20, 10, 7 and 5 copies input per reaction: each in 10 replicates. The data were plotted and analyzed according to the routine analyses provided in the software package with the ABI 7500 SDS instrument. (E) Instead of measuring 60 ddPCR replicates to obtain 95% confidence, we obtained an approximate estimation of the LoD using a lower number of ddPCR reaction replicates and required the false negative rate to be below 5% (i.e. all 10 replicates have to be positive).

    Article Snippet: In conclusion, combining two ddPCR assays for SIV nucleic acids detection with the RainDance ddPCR platform can enable a large amount of input DNA to be analyzed per reaction, and can overcome severe RNA inhibition when combined with suitable reverse transcription enzyme(s).

    Techniques: Generated, Real-time Polymerase Chain Reaction, Software

    ddPCR and qPCR comparison. (A) Sample inhibition comparison between ddPCR and qPCR. An ovarian DNA sample (Rhesus macaque 311–08) was subjected to nested (i.e. preamplified) qPCR analysis or direct RainDance (“RD” in the table header) ddPCR analysis for SIV DNA viral load. At 3.7 million cell input per reaction (at the preamp step), SIV qPCR signal is inhibited by 99%, while at 4 M cell input per direct ddPCR reaction, SIV signal is not inhibited. Note that in the qPCR graph, the data point (32.6 ± 5.4) on the left (0.37 million cell input per reaction) was derived from a standard curve, while the data point (4.0) on the right (3.7 million cell input per reaction) was derived from Poisson statistics (i.e. DNA copies derived from the positive well rate among the replicate reactions). (B) Quantification of Rhesus macaque necropsy tissue DNA samples (from Rhesus macaque 27882) (including an uninfected negative control sample) for SIV DNA using ddPCR. (C) Comparison between ddPCR and nested qPCR analysis results for the necropsy tissue samples in (B). RD, RainDance. S.D., standard deviation.

    Journal: PLoS ONE

    Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

    doi: 10.1371/journal.pone.0233085

    Figure Lengend Snippet: ddPCR and qPCR comparison. (A) Sample inhibition comparison between ddPCR and qPCR. An ovarian DNA sample (Rhesus macaque 311–08) was subjected to nested (i.e. preamplified) qPCR analysis or direct RainDance (“RD” in the table header) ddPCR analysis for SIV DNA viral load. At 3.7 million cell input per reaction (at the preamp step), SIV qPCR signal is inhibited by 99%, while at 4 M cell input per direct ddPCR reaction, SIV signal is not inhibited. Note that in the qPCR graph, the data point (32.6 ± 5.4) on the left (0.37 million cell input per reaction) was derived from a standard curve, while the data point (4.0) on the right (3.7 million cell input per reaction) was derived from Poisson statistics (i.e. DNA copies derived from the positive well rate among the replicate reactions). (B) Quantification of Rhesus macaque necropsy tissue DNA samples (from Rhesus macaque 27882) (including an uninfected negative control sample) for SIV DNA using ddPCR. (C) Comparison between ddPCR and nested qPCR analysis results for the necropsy tissue samples in (B). RD, RainDance. S.D., standard deviation.

    Article Snippet: In conclusion, combining two ddPCR assays for SIV nucleic acids detection with the RainDance ddPCR platform can enable a large amount of input DNA to be analyzed per reaction, and can overcome severe RNA inhibition when combined with suitable reverse transcription enzyme(s).

    Techniques: Real-time Polymerase Chain Reaction, Inhibition, Derivative Assay, Negative Control, Standard Deviation

    Identifying an optimal ddPCR assay format and condition. Performance of an MGB probe-based SIV gag ddPCR assay using TaqMan genotyping mastermix on (A) SIV (on CCR5 background) spike-in templates and (B) ovary tissue DNA from a Rhesus macaque (311–08) infected with SIVmac239. (C) A summary of all other tested conditions (probe and mastermix) and the observed issue(s) associated with each condition.

    Journal: PLoS ONE

    Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

    doi: 10.1371/journal.pone.0233085

    Figure Lengend Snippet: Identifying an optimal ddPCR assay format and condition. Performance of an MGB probe-based SIV gag ddPCR assay using TaqMan genotyping mastermix on (A) SIV (on CCR5 background) spike-in templates and (B) ovary tissue DNA from a Rhesus macaque (311–08) infected with SIVmac239. (C) A summary of all other tested conditions (probe and mastermix) and the observed issue(s) associated with each condition.

    Article Snippet: In conclusion, combining two ddPCR assays for SIV nucleic acids detection with the RainDance ddPCR platform can enable a large amount of input DNA to be analyzed per reaction, and can overcome severe RNA inhibition when combined with suitable reverse transcription enzyme(s).

    Techniques: Infection

    SIV ddPCR DNA assay performance. (A) SIV ddPCR DNA assay detection of low single digit level SIV DNA input and linear dynamic range of the SIV ddPCR DNA assay. Different amount of SIV DNA template was spiked in 1 million Rhesus macaque PBMC equivalent of genomic DNA background each. SIV DNA amount was quantified by direct ddPCR. (B) Quantification data of Fig 5A.

    Journal: PLoS ONE

    Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

    doi: 10.1371/journal.pone.0233085

    Figure Lengend Snippet: SIV ddPCR DNA assay performance. (A) SIV ddPCR DNA assay detection of low single digit level SIV DNA input and linear dynamic range of the SIV ddPCR DNA assay. Different amount of SIV DNA template was spiked in 1 million Rhesus macaque PBMC equivalent of genomic DNA background each. SIV DNA amount was quantified by direct ddPCR. (B) Quantification data of Fig 5A.

    Article Snippet: In conclusion, combining two ddPCR assays for SIV nucleic acids detection with the RainDance ddPCR platform can enable a large amount of input DNA to be analyzed per reaction, and can overcome severe RNA inhibition when combined with suitable reverse transcription enzyme(s).

    Techniques:

    Tissue DNA inhibition for SIV viral quantification. DNA was extracted from 7 tissues from a necropsied Rhesus macaque (310–08) that was infected with SIVmac239 (see Methods section for details). DNA samples (undiluted or 1:10 diluted) were first subject to preamplification, and 10% of each preamp product was subject to duplex qPCR of Rhesus macaque SIV gag and CCR5 [ 14 ]. 1000 copies of SIV DNA standard was spiked into one reaction containing the preamp product to assess PCR inhibition in tissue DNA, while a buffer spike (1000 copies of SIV DNA standard spiked into buffer) served as “no inhibition” control. SIV gag spike copy (lower left) for each sample was derived from the SIV gag spike Ct (upper left; compared to buffer spike Ct), while CCR5 average copies (single stranded) were derived from a CCR5 standard curve (upper right). In addition, the CCR5 copies (single stranded) were independently determined from the DNA input into each reaction (lower right) based on DNA concentration measurement using NanoDrop.

    Journal: PLoS ONE

    Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

    doi: 10.1371/journal.pone.0233085

    Figure Lengend Snippet: Tissue DNA inhibition for SIV viral quantification. DNA was extracted from 7 tissues from a necropsied Rhesus macaque (310–08) that was infected with SIVmac239 (see Methods section for details). DNA samples (undiluted or 1:10 diluted) were first subject to preamplification, and 10% of each preamp product was subject to duplex qPCR of Rhesus macaque SIV gag and CCR5 [ 14 ]. 1000 copies of SIV DNA standard was spiked into one reaction containing the preamp product to assess PCR inhibition in tissue DNA, while a buffer spike (1000 copies of SIV DNA standard spiked into buffer) served as “no inhibition” control. SIV gag spike copy (lower left) for each sample was derived from the SIV gag spike Ct (upper left; compared to buffer spike Ct), while CCR5 average copies (single stranded) were derived from a CCR5 standard curve (upper right). In addition, the CCR5 copies (single stranded) were independently determined from the DNA input into each reaction (lower right) based on DNA concentration measurement using NanoDrop.

    Article Snippet: On a per reaction basis, the ddPCR DNA assay can tolerate significantly more DNA input compared to qPCR or Bio-Rad ddPCR platforms.

    Techniques: Inhibition, Infection, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Derivative Assay, Concentration Assay