One-step reverse transcription-PCR (RT-PCR) is widely used in RNA analysis, such as genome expression, virus detection, and many fields from life science research to diagnosis. One-step Crystal RT-Digital PCR enables direct detection of multiple target genes (internal reference genes, multiple target genes) and viral RNA in RNA samples without the need for a standard curve, while enabling accurate detection of subtle differences in gene expression.
One-step Crystal RT-DPCR (Crystal RT-dPCR) enables the same reaction to perform three different mRNA analyses through three fluorescent channels, which can significantly and accurately distinguish 1.5 times the difference in gene expression (Figure 1). .
Figure 1: One-step Crystal RT-DPCR (Crystal RT-dPCR) Absolute quantification of mRNAs in human total RNA 1.5-fold dilution of human total RNA, ranging from 0.06 ng/μl to 3.6 ng/μl. Three-step absolute quantitative detection of three mRNAs of GUSB, ALB and TSN was performed by one-step Crystal RT-digital PCR. Using 1x qscript XLT One-step RT PCR Toughmix® and three kinds TaqMan TM probes FAM-, HEX and Cy5- labeled. The results of three repeated experiments showed that each target gene has good linearity, accuracy and reproducibility. GUSB = 588. 8 cp / ng: ALB = 96.5 cp / ng: TSN = 980.6 cp / ng
Quantitative detection of low expression level transcripts <br> Very challenging for detection of low abundance transcripts in high background total RNA, using Crystal digital PCR technology for detection of low ratios of transcript quantitation for excellent linearity and reproducibility (Figure 2A). Even if the fluorescence intensity was consistent with a high concentration of total RNA background, the reaction efficiency was not affected (2 μg - Figure 2B).
Figure 2: Absolute quantification of mRNA in high-concentration total RNA by one-step Crystal RT-digital PCR Human total RNA was double-diluted and the concentration ranged from 2 μg to 125 ng. Absolute quantification of ALB mRNA was performed by one-step Crystal RT-digital PCR. Using 1x qscript XLT One-step RT PCR Toughmix® and HEX- labeled TaqMan TM probes.
The linear plot shows the accuracy and repeatability of the results of the repeated three experiments (A), and no change in fluorescence was observed in the droplet scatter plot (B). RFU: Relative fluorescence unit. * 27 μL per reaction.
Absolute quantification of viral RNA <br> The dengue type 1 virus DEN-1 and three other similar serotypes can pose fatal risks such as dengue hemorrhagic fever and dengue shock syndrome. One-step Crystal RT-Digital PCR enables absolute quantitative detection of dengue viral load in 2.5 hours without the need for standards and standard curves, fast, friendly and suitable for all laboratories and workflows.
One-step Crystal RT-DPCR (Crystal RT-dPCR) enables the same reaction to perform three different mRNA analyses through three fluorescent channels, which can significantly and accurately distinguish 1.5 times the difference in gene expression (Figure 1). .
Figure 1: One-step Crystal RT-DPCR (Crystal RT-dPCR) Absolute quantification of mRNAs in human total RNA 1.5-fold dilution of human total RNA, ranging from 0.06 ng/μl to 3.6 ng/μl. Three-step absolute quantitative detection of three mRNAs of GUSB, ALB and TSN was performed by one-step Crystal RT-digital PCR. Using 1x qscript XLT One-step RT PCR Toughmix® and three kinds TaqMan TM probes FAM-, HEX and Cy5- labeled. The results of three repeated experiments showed that each target gene has good linearity, accuracy and reproducibility. GUSB = 588. 8 cp / ng: ALB = 96.5 cp / ng: TSN = 980.6 cp / ng Quantitative detection of low expression level transcripts <br> Very challenging for detection of low abundance transcripts in high background total RNA, using Crystal digital PCR technology for detection of low ratios of transcript quantitation for excellent linearity and reproducibility (Figure 2A). Even if the fluorescence intensity was consistent with a high concentration of total RNA background, the reaction efficiency was not affected (2 μg - Figure 2B).
Figure 2: Absolute quantification of mRNA in high-concentration total RNA by one-step Crystal RT-digital PCR The linear plot shows the accuracy and repeatability of the results of the repeated three experiments (A), and no change in fluorescence was observed in the droplet scatter plot (B). RFU: Relative fluorescence unit. * 27 μL per reaction.
Absolute quantification of viral RNA <br> The dengue type 1 virus DEN-1 and three other similar serotypes can pose fatal risks such as dengue hemorrhagic fever and dengue shock syndrome. One-step Crystal RT-Digital PCR enables absolute quantitative detection of dengue viral load in 2.5 hours without the need for standards and standard curves, fast, friendly and suitable for all laboratories and workflows.
Figure 3: One-Step Crystal RT-Digital PCR Absolute Quantification of DEN-1 Viral Load
DEN-1 viral RNA (manufacturer quantified by digital PCR) was spiked with 300 cp human total RNA as background and ABL mRNA as internal reference. The DEN-1 virus was detected using a 1x qscript XLT One-step RT PCR Toughmix®, and the ABL gene was detected by a HEX probe. The linear fit is 0.99 and the repeatability is good.
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