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Differential Gene Expression Using Digital PCR
Detect low-fold changes in gene expression
Real-time PCR is commonly used to detect differential gene expression; however, this approach is generally limited to detecting changes that vary by two-fold or more. For some studies, detection of expression changes less than two-fold may be required. Furthermore, it is often necessary to express differential gene expression with respect to a reference gene, such as a housekeeping gene like actin.
With the ability to achieve highly precise measurements of ±10% or better, digital PCR is capable of resolving changes of two-fold or less.
In addition, the ability of digital PCR to determine absolute quantification of a transcript obviates the need for a reference gene. Like real-time PCR, digital PCR requires the conversion of RNA to cDNA. Since the efficiency of conversion is important to experimental sensitivity, we offer the High-Capacity cDNA Reverse Transcription Kit, which seamlessly integrates into your digital PCR gene expression workflow.
Figure 1.Quantitation precision comparison between digital PCR (A) and real-time qPCR (B). Quantitation precision comparison between digital PCR (top) and real-time qPCR (bottom). Sample 1 through 5 is a mixture of synthetic miRNA of hsa-miR-19b and hsa-miR-92 at different ratio: sample 1, 100%; sample 2, 95%; sample 3, 90%; sample 4, 75%; sample 5, 50%. After reverse transcription, cDNA was run with qPCR and digital PCR with QuantStudio 3D system. Delta Ct of qPCR between hsa-miR-19b and hsa-miR-92 were reported for each sample. The relative quantitations for digital PCR results were reported in percentile for each sample. Digital PCR with QuantStudio 3D is able to discriminate a 5% difference between sample 1 and 2 (indicated by non-overlapping circles by Tukey-Kramer HSD test), while real-time qPCR was not able to discriminate even 10% difference between sample 1 and sample 3. Tukey-Kramer HSD test was done within JMP software with experiment replicates.
Literature
Poster: Precise and Accurate Determination of MicroRNA Precursors by Digital PCR
Product Bulletin: QuantStudio™ 3D Digital PCR System
User Guide: QuantStudio™ 3D Digital PCR System
Digital PCR Experiment Design Guide
QuantStudio™ 3D Digital PCR System
Absolute quantification, using sealed-chip technology, for a reliable method and precise, sensitive data.
For Research Use Only. Not for use in diagnostic procedures.