How do molecular biology services work?
KCAS Bio’s purpose-built molecular services combine qPCR, ddPCR within a controlled, unidirectional workflow system to deliver high-quality, contamination-free analysis.
Our molecular biology workflow
Our purpose-built molecular facility enables a controlled, unidirectional workflow designed for high-quality PCR and genomic analyses.
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Sample Pre-ProcessingSamples enter for any pre-processing steps: Homogenization, tissue disruption, fractionation
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Nucleic Acid ExtractionDedicated space for sample extraction using QIACube Connect Mdx | Maxwell RSC | Qubit Flex Fluorometer | NanoDrop Eight
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Master MixReaction mixes without nucleic acid are prepared and distributed into 96-well plates Both ddPCR and qPCR workflows | PCR workstations
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Template AdditionNucleic acid templates extracted are added to reaction plates that were prepared ddPCR reactions undergo droplet generation on the AutoDG
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PCR AmplificationComplete reaction plates undergo PCR amplification and analysis QuantStudio 7 Pro with SAE Console | QX200 driven by QX Manager Software Regulatory Edition
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Post-AmplificationDedicated space for analysis of PCR products Future capabilities to include Sanger sequencing and NGS
qPCR Overview
Quantitative PCR (qPCR) is a modification of PCR in which a fluorescent dye is added to the reaction to detect the amount of product present at the end of each heating/cooling cycle.
- qPCR can be performed on cDNA synthesized from RNA for RT-qPCR
- New copies of DNA are detected in “real-time”
- Number of copies of target in starting sample determined by standard curve
- Relative quantitation compares relative fluorescence between samples for target gene and normalization gene
dPCR Overview
Digital PCR (dPCR) is a modification of PCR in which reactions are partitioned into thousands of nanoliter-sized reactions.
- dPCR can be performed on cDNA synthesized from RNA for RT-qPCR or RT-dPCR
- Fluorescent signal is measured in each partition with a binary (“digital”) readout of yes or no
- Poisson statistics are used to determine the number of copies of target in the starting sample from the number of positive and negative partitions
Droplet digital PCR (ddPCR) PCR reaction is partitioned into oil in water droplets.
qPCR vs dPCR
|
Feature |
qPCR |
dPCR |
|---|---|---|
|
Quantification |
Uses standards for measurement |
Absolute measurement without standards |
|
Sensitivity |
High |
Ultra-high |
|
Precision |
Reliable |
Highly precise |
|
Low Copy Detection |
Good |
Excellent |
|
Copy Number Analysis |
Suitable |
Ideal |
|
Inhibitor Tolerance |
Moderate |
High |
|
Turn around Time |
Fast |
Slightly longer |
|
Best For |
Routine analysis and screening |
Ideal for rare sequence detection, low-copy targets, or when only limited amounts of input material are available |
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