Regarding digital PCR, please keep these dry goods [experience comment]

Compared with traditional qPCR, digital PCR (dPCR) is a highly sensitive existence that can achieve absolute quantification of nucleic acids and detection of rare alleles

For more than 20 years, digital PCR technology has been continuously improved and has been applied in many aspects, including quantifying cancer markers, measuring viral load, distinguishing genome mutations, verifying the results of next-generation sequencing, gene expression analysis, transgene detection, and environmental monitoring, etc.

Sample Preparation

How much starting DNA to use

The starting amount of DNA will depend on the digital PCR system you are using

Concentrate FFPE samples before amplification

When dealing with highly degraded DNA, such as FFPE samples, there may be a big difference between quantifiable DNA and amplifiable intact DNA

experimental design

Reduce heating and cooling rate

Nowadays, the heating and cooling rate of thermal cyclers is staggering

Modify PCR amplification conditions

When dealing with tricky templates, PCR cycle conditions can be changed

• For longer amplicons (> 400 bp), change the two-step or three-step operation and add a 72°C extension cycle for 1-6 minutes, depending on the length of the amplicon

data analysis

Looking for real positive samples

In order to determine whether a sample is truly positive, Bio-Rad has a good suggestion

When detecting rare DNA, 7-10 μg of DNA may be required

Understanding error bars

In the ddPCR system, the error bar for each well represents a 95% confidence interval

More suggestions

Digest the sample

When the amount of gDNA exceeds 66 ng, it is recommended to use restriction enzymes for digestion

Digestion can be carried out directly in the ddPCR mixture

Use multiple reference genes

When evaluating copy number variation (CNV), especially cancer samples, it is necessary to ensure that the reference test is stable at 2 copies per genome

Concluding remarks

Nowadays, the scientific community requires higher data accuracy and more reliable results.

In-depth understanding of Bio-Rad's droplet digital PCR system