Question 1

What is miniaturization?

Accepted Answer

Miniaturization is the term for scaling down the volume of a reaction mixture or assay in molecular biology. Miniaturization is useful for assay development, design of experiment (DoE) runs, and high-throughput screening. As research now shifts to single cell applications, miniaturization can help do a lot more with a single sample, given the low input volume.

Question 2

How does miniaturization work?

Accepted Answer

Scaling down common molecular biology and genomics protocols is simple because these applications use active enzymes that are both sensitive and precise, making them are highly amenable to scaling down the working volume to 1/10th of the prescribed volume or even lower (though this may be restricted by the kit being used).

At such low volumes, miniaturization of protocols must take advantage automated liquid handling systems. Liquid handlers can perform standard to high throughput execution of protocols, reduce overall costs, and increase data quality through automated sample tracking. Automation also helps reliability by removing human error from the equation, especially for mentally tiring manual processes, and allows the researcher to spend more time on research, data handling, and analysis.

Question 3

Why is miniaturization important?

Accepted Answer

In addition to the obvious savings on reagent, miniaturization uses less of the sample in question. This allows the researcher to gain more data points from a single sample, allowing more strategic use of further assays.

Strategic use of miniaturization proved useful during the COVID-19 pandemic as supply chains of key diagnostic reagents became scarce, forcing clinicians to consider scaling down approved protocols.

In our experience, miniaturization reduces experimental cost at least by 75% while preserving cell/library success rate and method sensitivity.

Other main advantages to miniaturization are:

High resolution genomic (including single cell) and NGS libraries
Genomics work with less reagent and sample
Scaling of an assay from low throughput to high throughput
Processing more cells in fewer batches
Automated liquid handling adds benefits such as:

Reduced hands-on time in lab
Increased pipetting accuracy
Reduced user variability
Less pipetting strain on the researcher
Minimized cross contamination
These factors are good to keep in mind when starting your miniaturization and automation journey, as you can build your vision of a lab of the future today.

Question 4

What are the advantages of automation?

Accepted Answer

Over the years, multi-well plates have been condensed to fit more wells. The 96- and 384-well plates are common, however 1536-well plates are also in use. These are difficult to handle with multiple reagents, even with electronic pipettes. Additionally, these manual handling tasks are a poor use of a skilled researcher’s brain power. More and more labs are moving towards automated liquid handling due to the immediate benefits of reliability and accuracy in carrying out experiments, but also increasing the complexity of the experiments that can be done while freeing up the researcher to focus on experimental design and data analysis.

Question 5

What throughput of samples do you require?

Accepted Answer

When we talk about high-throughput screening (HTS) and ultra-high-throughput screening (uHTS), this means samples are processed on an order of up to 100,000 per day for HTS and higher in the case of uHTS. Many genomics labs use medium to high throughput for DNA library screenings or whole genome sequencing projects.

Question 6

What volume range do you require?

Accepted Answer

The volume range and the way in which liquid is handled are inextricably linked. When miniaturizing protocols, you will require a specialist liquid handler operating a pipetting range in the nano liter – micro liter (nL – μL) range. More standard automated liquid handling platforms operate in the micro liter to milliliter (μL – mL) range.

How does the device handle liquid?

Platforms that use air displacement pipettes, like how a standard hand-held pipette works, can be disturbed by air pressure, and may require additional calibration when aspirating and dispensing viscous reagents, such as those containing glycerol or volatile reagents such as Ethanol.

Platforms that use positive displacement tips, where a plunger comes in direct contact with the reagent, are unaffected by viscosity and air pressure. This style of dispensing is suited to additive kit protocols and uses a lower dead volume of reagent than others. If repeat dispensing small volumes at a time from a larger tip to multiple wells, the same tip cannot be used to mix by pipetting. This is not an issue in miniaturized protocols however, as homogenization of the sample occurs by turbulent mixing and diffusion.

Acoustic liquid handlers, as the name suggests, use sound to transfer precision droplets of liquid to a target well. Like air displacement pipettes, these need to be calibrated to the viscosity of the reagents. By not using plastic tips, these devices significantly reduce the amount of waste produced by a lab. However, they are unable to transfer ‘intra-plate’ or handle higher transfer volume as they rely on an inverted destination plate positioned directly over the source plate which can be limiting for some applications.

Always ask vendors to provide full information on the range, variance, and accuracy of their pipetting/dispensing technology , across a range of representative liquids matched to your application, to ensure it fits with the needs of your miniaturized protocol.

Question 7

What is the footprint of the device?

Accepted Answer

It may seem obvious to check the footprint of a new piece of lab kit, but with robotics it is equally important to be aware of any moving parts or additional space that may be required when installing. A tabletop unit may fit your bench perfectly but if it also advises a specified clearance between the device and wall, it may compromise safety and not be a good fit.

Question 8

Is specialized knowledge required to operate this platform?

Accepted Answer

A major barrier to adopting automation – if not the biggest barrier - is the worry that specialized knowledge is required to operate the device. Automated liquid handling is, for the most part, rather straightforward now. Several automated platforms are available as ‘plug and play’ and require little or no software coding expertise. Vendors typically provide training and may assist with protocol development. Additionally, find out if a copy of the software is freely available to install on other computers. This will allow you to design experiments away from the lab, and simply upload the file when you get in – a real advantage when stuck working from home!

Question 9

What consumables will you require and how secure is the supply chain?

Accepted Answer

With automation comes a big change to your supply chain. If the COVID-19 pandemic has taught us anything in science, it is to be mindful of where our consumables and reagents originate and if they will be delayed. With strategic implementation of miniaturization and automation in your lab, you can regain control of your needs and better cope with supply chain issues in the future.

Question 10

How much can the reaction volumes be reduced?

Accepted Answer

This depends on the kit chemistry and sample type. Some NGS kits are robust at a wide range of volumes and sample concentrations, while others allow up to a 4-fold volume reduction. Sample type (e.g. single cells vs bulk DNA/RNA, or high vs low RNA content) should also be considered. Generally single cell workflows allow substantial miniaturization as the input is just a few picograms of genetic material. In the case of bulk RNA and DNA miniaturization, it is still possible but should be less extreme to ensure sufficient coverage and depth of sequence data. Samples for bacterial and viral genomic amplicons, due to their low complexity, can be processed using low volumes of the reagents.

Question 11

Are accuracy and precision compromised at low volumes?

Accepted Answer

Yes there is a risk associated when reducing volumes unless you consider using a fit for purpose system. For precision liquid handling at very low volumes, automated liquid handlers like the mosquito® HV genomics and dragonfly® discovery use true positive displacement technology. Here, the plunger comes in direct contact with the reagent and liquid dispensing is unaffected by viscosity and air pressure. This style of dispensing is suited to additive kit protocols and uses a lower dead volume of reagent than others.

Question 12

Is there a risk of contamination?

Accepted Answer

For sample transfer, discrete disposable tips should be used per sample. For reagent additions non-contact dispensing is a good option as this typically utilizes disposable sterile tips which do not contact the target plate or tube. They also dispense liquid without splashing. If contamination is a major concern, check if it is possible to place the liquid handler in a laminar flow hood.

Question 13

What is the impact of evaporation on results?

Accepted Answer

Speed of reagent dispensing is important. Consider a humidity chamber and/or cooling modules to decrease the evaporation rate. Evaporation will always be slower in a smaller diameter well, so miniaturized reactions may work better in 384 well plate than in a 96 well plate. In other words, the smaller the better!

Question 14

What is the reagent waste due to dead volume?

Accepted Answer

Assuming positive displacement technology is utilized for both pipetting and dispensing steps in miniaturized workflows, the amount of dead volume depends more on the labware chosen for the tips to fill from. Choose labware with low dead volume plates or reservoirs.

Question 15

How much plastic waste will miniaturized/automated liquid handling produce?

Accepted Answer

Automation reduces human error by precise execution of protocols, minimizing unnecessary repetition. Technologies like positive displacement pipette tips do not physically contact biological samples when dispensing reagent and can be disposed of in an eco-friendly manner. When miniaturizing protocols, a single tip can dispense multiple volumes, thereby reducing the number of tips required for a given process. As mentioned in the previous section, acoustic liquid handling platforms do not require tips at all, although they require a specific type of source plate the samples or reagents to be dispensed need to be loaded in to.

Question 16

Will adopting miniaturized liquid handling require additional investment?

Accepted Answer

Switching from 96 well plate to more compact 384 well format requires a thermocycler with an adequate heating block. You may simply need to buy the required block though some instruments may not be compatible, and a new thermocycler must be purchased.

Question 17

What is the overall cost of running an experiment?

Accepted Answer

While it might be possible to reduce reagent cost, if consumables (plates, tips) are expensive then total savings can be much lower than expected. This has prompted a shift towards more compact plates, such as 384 well plates.

Question 18

Is specialist knowledge required? How much reliance on vendor tech support?

Accepted Answer

Automated liquid handling devices nowadays typically have user friendly software, are easy to program (no programming skills required) and are beneficial, especially in an academic environment. Getting the most from your technology may extend beyond simple technical support but also from by Field Application Scientists that provide scientific advice and assist laboratory teams in the installation of instruments and design of scientific applications. As each vendor and device is different, do check before purchase if any specialist knowledge is required, what tech support comes with the package, details of license agreements, and of course training.

Question 19

Will these instruments become redundant in future?

Accepted Answer

Ideally not in our lifetime! Most devices are designed with an open platform that allows them to be flexible and change direction if needed.

Question 20

What is genomics?

Accepted Answer

Genomics is the study of genomes covering everything from their structure and function to their editing and evolution. Most genomics techniques are amenable to miniaturization because they use enzymatic reactions and genetic material samples that can be efficiently scaled down.

Question 21

How does genomics work?

Accepted Answer

Genomics is an interdisciplinary field using advancements in molecular biology tools to assess the makeup of a genome, the transcription of its genes, and more. Common techniques used include CRISPR, NGS, PCR and quantitative reverse transcription PCR (RT-qPCR), as well as transcriptomic techniques such as DNA-seq and RNA-seq.

Question 22

Why is genomics important?

Accepted Answer

PCR and NGS revolutionized diagnostics and public health as advancements in genomic technology are helping usher in the era of personalized medicine, better diagnostic techniques, and new therapeutics.

NGS is powering precision technologies like single cell sequencing, keeping track of mutations accumulating in SARS-CoV-2 during the COVID pandemic, and creating masses of high-quality data for artificial intelligence platforms to trawl. These data are now playing a role in population genomics and conservation.

Genomics is also crucial for the emerging field of synthetic biology/engineering biology in identifying and engineering novel metabolic pathways. These can be used for producing bio-based sources of currently unsustainable or petrochemical-derived compounds, antimicrobial compounds, and therapeutics.

Question 23

What is PCR?

Accepted Answer

PCR makes copies of a piece of DNA in a matter of hours which can then be used in cloning, sequencing, or simply just run on a gel and viewed. RT-qPCR and Real Time PCR (qPCR) are used to quantify gene copy numbers, with practical application in diagnostics such as Sars-CoV-2 detection. RT-qPCR relates to quantification of gene expression as the reverse transcriptase step creates the DNA template from RNA.

Question 24

How does PCR work?

Accepted Answer

PCR works in a stepwise process: Denaturation of DNA; Annealing of oligonucleotide primers; and Extension of a new DNA strand with DNA polymerase. This process is repeated, typically in 25 or more cycles. Primers and nucleotides in the mixtures can be modified to add features such as restriction enzyme sites or fluorescent tags. RT-qPCR and qPCR uses the same method, but additionally use fluorescence to quantify DNA amplification as it happens, so there is no need for a gel run.

Question 25

Why is PCR important?

Accepted Answer

The PCR mixture is straightforward and entirely additive, lending itself to easy automation. RT-qPCR is best suited, as the fluorescence detection of amplification gives an immediate result, whereas a small amount of amplified DNA from a standard PCR in miniaturized form may not yield enough volume to visualize on an electrophoresis gel. In functional genomics, PCR is used to create recombinant DNA – typically a gene of interest on a plasmid vector – for expression in a cell.

Question 26

Benefits of miniaturizing PCR

Accepted Answer

PCR is the backbone of molecular biology. Its applications in biotechnology, therapeutics development and diagnostics make it an essential tool for every life science researcher. The technique is highly sensitive, requiring only picograms of DNA template in order to work, and is therefore highly amenable to miniaturization. Miniaturizing PCR for diagnostics can help stretch reagent use and reduce costs of diagnostic tests such as for COVID-19.

Question 27

What is molecular cloning and vector assembly?

Accepted Answer

Plasmid vectors are assembled (and disassembled) using a variety of methods, including type II restriction enzymes, Golden Gate assembly, and Gibson assembly. Using PCR amplicons, molecular parts such as promoter, replicons, transposons, and terminators, plasmid vector backbones can be built with gene circuits and properties to study functional and structural genomics of an organism.

Question 28

How does molecular cloning and vector assembly work?

Accepted Answer

Cloning and vector assembly commonly uses type II restriction enzymes. Restriction enzyme recognition sites are straightforward to add onto PCR primers, making DNA fragments simple to clone into a vector.

Golden Gate assembly uses these enzymes sequentially. A vector of multiple parts can be assembled in a one-pot reaction. The major detraction of using type II restriction enzyme sites is that they tend to leave ‘scars’ or traces of themselves in the backbone of the plasmid vector or DNA sequence, thus shifting open reading frames or putting added distance between a gene and its promoter sequence.

Gibson assembly allows for scarless cloning. This technique makes use of DNA’s ability to anneal to itself. Fragments of the vector and insert are designed with approximately 20-40 bp DNA overlapping with the fragment next to it. Three enzymes are required to complete the cloning: A 5’ exonuclease is added to pare back one strand of DNA on each side, allowing them to fit together; DNA polymerase to close the gaps after DNA has annealed; DNA ligase to seal the nicks.

Question 29

Why is molecular cloning and vector assembly important?

Accepted Answer

The importance of the ability to introduce DNA to an organism cannot be overstated. From functional genomics to development of gene therapies, introducing DNA – either introducing a new gene product, overproduction of an existing gene product, or to delete, silence or interfere with existing genes – is fundamentally essential to the field of genomics.

Question 30

Benefits to miniaturizing molecular cloning and vector assembly

Accepted Answer

Restriction enzyme reactions, Golden Gate, and Gibson Assemblies can be performed in one-pot with incubation steps, making them favorable cloning techniques among those who use automation. Both methods are suitable for miniaturization. This can help reduce cost by minimizing reagents and save time on library preparation.

Question 31

What is next generation sequencing?

Accepted Answer

NGS is a rapid DNA sequencing method that is useful for reading whole or partial genomes, confirming DNA sequence of PCR amplicons, and can be used in diagnostics. The technique can also be applied to cDNA and RNA, resulting in rapid and accurate transcriptomic analyses.

Question 32

How does next generation sequencing work?

Accepted Answer

Next generation sequencing works like PCR but uses fluorescent di-nucleotide phosphate bases (dNTPs). During the extension step, DNA polymerase incorporates fluorescent dNTPs which emit a signal. This signal is picked up by a detector and records the DNA sequence in real time.

Question 33

Why is next generation sequencing important?

Accepted Answer

NGS is commonly applied to whole genome sequencing due to its speed but is also used for smaller fragments such as PCR products and plasmid vectors, lending itself to every area of biological research. It is also used to monitor gene expression from cDNA generated from RNA, allowing researchers to take a snapshot of gene expression at a particular time in a cell. This method is called RNA-seq and has replaced the less reliable technologies of hybridization microarrays. Its sensitivity even allows single cell applications in the above. NGS can also be used in population genomics and diagnostics.

Question 34

How to prepare NGS libraries

Accepted Answer

To prepare a library for NGS, you must first consider the procedure they will be used for. The protocol below can be miniaturized to a final volume of 4 µL. Check out this demo of library preparation.

Question 35

Benefits of miniaturizing next generation sequencing

Accepted Answer

Because NGS is based on the principles of RT-qPCR (elongation and fluorescence) and the protocol is simple additive, it is easily miniaturized. This saves on very costly reagents (polymerase enzyme, buffers, fluorescent dNTPs) which currently stand in the way of achieving the $100 genome using NGS technology.

Question 36

What is spatial transcriptomics?

Accepted Answer

A major limitation of using single cell sequencing to study gene expression across a tumor is, after biopsy, it can be difficult to trace the single cell analyzed back to its precise location on the tissue. Spatial transcriptomics is a method of mapping gene activity across a tissue. This differs significantly from bulk RNA-seq which just an average across the whole sample

Question 37

How does Spatial Transcriptomics work?

Accepted Answer

A tissue sample is placed on a glass slide or in a plate arrayed with oligonucleotides containing positional barcodes and cDNA primers to capture mRNA from each position. The tissue is fixed and permeabilized, allowing capture of the mRNA. The captured fragments are converted to cDNA and sequenced using NGS and the positional barcodes allows for mapping back to the point of origin.

Question 38

Why is spatial transcriptomics important?

Accepted Answer

Spatial knowledge – physically where in the tissue the cell originated – is often lost in single cell RNA-seq. To get a better idea of how gene expression occurs in a dynamic, spatially organized tissue such as the liver, spatial transcriptomics is required to visualize and gain a quantitative analysis of the transcriptome in tissue samples. Spatial transcriptomic data is more resolved and can detect DNA perturbations on the transcriptomic level, allowing more accurate diagnoses.

Question 39

Benefits of miniaturizing spatial transcriptomics

Accepted Answer

As with other forms of single cell sequencing, miniaturization of spatial transcriptomics using automated liquid handling results in robust, reliable execution of the protocol while saving on expensive and sensitive reagents and making use of small sample volumes.
It should be noted, miniaturization can only be performed on a plate (as opposed to on-chip methods) and is dependent of the volume in which the sample has been added to the plate in question.

Question 40

What is CRISPR?

Accepted Answer

Clustered regularly interspaced short palindromic repeats (CRISPR) is a family of bacterial DNA-editing proteins that effectively acts as the microbial immune system. Genetic editing is a useful technique in functional genomics. It allows the researcher to add or remove from the genome regions suspected to be involved in regulation of gene expression, as well as alter the location of genes on chromosomes to observe the effect location has on gene activity.

Question 41

How does CRISPR work?

Accepted Answer

When a bacterium encounters a bacteriophage (a virus for bacteria), CRISPR proteins may recognize its DNA, physically chop it up, and insert parts of it into the bacterial genome so it can recognize it better in the future. This bacterial DNA editing mechanism works in other cell types, including humans. The CRISPR proteins can be re-targeted using a special type of RNA called guide RNA (crRNA) allowing precision modifications to be made anywhere in any genome. Once the CRISPR-Cas complex is guided to the target site, it makes a nick in the DNA. Then, the host organism fixes the nick with its own DNA repair mechanism. This is where another piece of DNA matching upstream and downstream of the nick can recombine into the genome, via a process called homologous recombination.

Question 42

Why is CRISPR important?

Accepted Answer

CRISPR has many applications in R&D gene editing, knocking in and knocking out DNA. One recent example in healthcare genomics, CRISPR allows for rapid in vivo functional genomics in patient-derived xenografts where tumor tissue is transplanted into a mouse model. Recent research has shown that CRISPR can perform targeted gene disruption, allowing clinicians to observe genetic dependencies within the tumor and analyze mechanisms of acquired drug resistance by site-specific editing.

Question 43

Benefits to miniaturizing CRISPR?

Accepted Answer

CRISPR effectiveness is often based on the target in question and may not always be effective. Large CRISPR libraries can be generated and screened with ease using automation and miniaturization to keep costs low which maintaining reliable results. Much like other vector assemblies (see Molecular Cloning), a CRISPR vector library with crRNA and DNA repair sequence would be amenable to miniaturization as the process is largely additive.

Question 44

What is synthetic biology/engineering biology?

Accepted Answer

Synthetic biology/engineering biology is a precision approach to biological research. Based on the Design-Build-Test-Learn framework, automation and software play a major role in carrying out experiments and providing robust and reliable data.

Question 45

How does synthetic biology/engineering biology work?

Accepted Answer

Much like how the silicon chip industry uses standardized parts to build its products, this field is trying to do similar. Standardized molecular parts – such as promoters, terminators, and riboswitches –make up the toolkit relied on in many protocols. Gene circuits, metabolic pathways and vectors are engineered, built, tested, and improved upon in an iterative design process.

Question 46

Why is synthetic biology/engineering biology important?

Accepted Answer

The field aims support a growing, sustainable bio-based economy through the development of new R&D tools, bio-based materials and products, and therapeutics. The next generation of bio-based products could be entirely synthesized from standardized DNA parts in the lab.

Question 47

Benefits to miniaturizing synthetic biology/engineering biology

Accepted Answer

Synthetic biology/engineering biology relies heavily on many of the NGS and genomics tools listed above and encourages the uptake of automation to ensure valid, reproducible results. Miniaturization helps by reducing environmentally hazardous reagent use and minimizing plastic waste, chiming with the bio-based philosophy driving the field forward. These techniques are highly amenable to miniaturization and, in carrying out high-throughput iterative cycles of experiments and vector/gene circuit building, may benefit from reduced costs by using miniaturized protocols.

The Why and How of Miniaturizing Genomics Applications

Contents

Introduction

Miniaturization and Automation