Here's an article that shows how (semi) quantitative digital droplet PCR can be done using a homemade microfluidic droplet prototyping toolkit. The authors detected and roughly quantified human GAPDH RNA transcripts using standard PCR laboratory equipment - very cool! - ThreadSky

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bento.bio • 98 days ago

Here's an article that shows how (semi) quantitative digital droplet PCR can be done using a homemade microfluidic droplet prototyping toolkit. The authors detected and roughly quantified human GAPDH RNA transcripts using standard PCR laboratory equipment - very cool!

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bento.bio•98 days ago

For reference: digital droplet PCR (ddPCR) is a quantitative method that splits a PCR into many tiny droplet “microreactors” containing a single DNA molecule (if appropriately diluted). Using fluorescence, successful amplicons are detected and counted to calculate the original target concentration.

bento.bio•98 days ago

The toolkit developed by Chen et al. (2023) was designed to be an adjustable, easy-to-use, and affordable prototyping system for a wide range of different microfluidic droplet applications, including ddPCR but also single-cell assays, drug assays, and antibiotic screening, among many others.

bento.bio•98 days ago

To be useful it needed to be affordable; to produce droplets of consistent and modifiable sizes; to have a platform to contain and visualise the droplets; and to allow easy imaging and counting. The authors’ solutions to these requirements included:

bento.bio•98 days ago

⭐A torq screwdriver tool to deform pipette tips so that they could produce droplets as small as 200 µM diameter

⭐A novel shallow-centre imaging surface made from double-sided tape or a 3D printed chip

⭐A microscope or smartphone imaging system to image droplets

bento.bio•98 days ago

As one test of their toolkit, Chen et al. (2023) created droplets as microreactors for reverse transcriptase ddPCR, aiming to quantify the concentration of human GAPDH RNA transcripts in their samples.

bento.bio•98 days ago

Although the method was not as accurate as a commercial ddPCR machine, they considered the method to be “sufficient for rough quantification and accurate detection for low-target concentration samples", which is impressive for a homemade kit!

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