User manual

Droplet Digital
™
PCR Applications Guide | 59
Gene Expression
The one-step RT-ddPCR kit for probes is formulated for efficient and sensitive reverse
transcription over a wide linear dynamic range of input RNA for ddPCR. Use a commercial
kit to extract RNA, then store RNA at 100 ng/µl in 1/10 TE buffer (0.1x TE) or other
appropriate buffer at –80˚C. A unique hot-start reverse transcriptase enables convenient
reaction setup. The reverse transcription reaction is performed at 55–60°C, enhancing the
specificity and efficiency of primer-mediated cDNA conversion. The thermostable enzymes
allow RNA template to be reverse transcribed and subsequently amplified in the same
reaction tube. Template-specific primer annealing at elevated temperatures significantly
improves stringency and melting of secondary structures.
Data Analysis
In QuantaSoft
™
software, create a plate map by designing a new plate with a layout
according to your experimental design. Designate the experiment type as Absolute
Quantification (ABS) in the Well Editor window during plate setup. Designate which assay is
the target and which is the reference assay (Channel 1 or 2) in the Well Editor window.
If the plate was set up for ABS analysis, automatic thresholding will determine concentrations
and populate the data tables in the analysis mode of the software. To get the ratio of target
over reference for gene expression, a duplex assay must be run with two different probes
(FAM and HEX [or VIC]).
In QuantaSoft data analysis, a button labeled Ratio will become active if the experiment
contains a duplex assay set up with a FAM assay and a HEX or VIC assay (Figure 6.1).
Clicking on the Ratio button plots the relative ratio of a/b (the concentration of the FAM
assay over the concentration of HEX or VIC assay). You can also click the Inverse button
(Figure 6.1) if you switched the assay dyes where FAM is the reference and HEX or VIC is
the target (the inverse makes the ratio b/a instead of a/b).
Fig. 6.1. QuantaSoft software ratio feature and inverse checkbox for gene expression ratios.