Real-time PCR

Real-Time (also called Quantitative-) PCR is based on the measurement of the accumulation of very short amplicons in real time. We currently propose two alternative detection methods.

• Sybr Green: a non-specific dye, which binds double-stranded DNA and fluoresces when intercalated.
• TaqMan probes: short oligonucleotides complementary to amplicons, which fluoresce during PCR (under degradation). TaqMan probes provide additional level of specificity compared to Sybr Green. Such probes can also be used for allelic discrimination by Q-PCR (one probe per allele is needed).

Real-Time PCR format

• 384-well plates

  Plates are processed on the QuantStudio 12K Flex systems from Thermo Fisher Scientific.
  This instruments is equipped with an automated device for plates loading. Three liquid handling robots (Tecan Freedom EVO) are included in the workflow for plates filling.
  This automatized station combines flexibility and high-precision measurement.

• OpenArrays

  OpenArrays are microscope slide–sized plates with 3’072 through-holes of 33 nL of volume, designed for Q-PCR. These assay-preloaded arrays are processed on the automated QuantStudio 12K Flex OpenArray AccuFill™ system (details in Thermo Fisher Scientific website).
  The OpenArray technology helps streamline genotyping and expression studies that use large numbers of samples, assays, or both. Custom and commercial panels are available.
  OpenArrays are probably the best-fit system for pharmacogenomics testing.

Applications

• Expression analysis, including miRNA detection
• Genotyping (= SNP detection = allelic discrimination)
• CNV analysis

Digital PCR

The aim of the Digital PCR is to quantify DNA molecules. It uses multiple PCR reaction partitions. Samples are diluted down to a limiting quantity such that most individual PCR reactions contain either zero or one target molecule. After PCR completion, the system counts the number of positive PCR wells that is the direct reflect of the number of molecules present.

The Platform is equipped with the QuantStudio 3D system from Thermo Fisher Scientific.

End point PCR using fluorescent-labeled probes (or Sybr Green) is done on chips containing 20K partitions. Chips are loaded on the ProFlex instrument. Imaging is done on the QuantStudio 3D.

Applications

• Absolute quantification (for DNA or cDNA)
• Copy Number Variation analysis
• Rare allele detection and quantification
• Low target copy detection