This research was made possible by the &#39;Flanders Resilience&#39; subsidy from the Flemish Government, originating from the &#39;European Recovery and Resilience Facility&#39; (RRF) (VV021/13). Figure 2 is created with Biorender.com.

The protocol described here is designed to quantify the viral genome titer of a purified, in-house produced AAV9 vector with green fluorescent protein (GFP) as the transgene12 with a high level of precision (Table 1). However, this protocol is applicable to any AAV serotype and any vector genome design, provided that the primer/probe sets are designed to target the specific vector genome of interest. Details of the reagents, primers, probes, and equipment used in this study are listed in the Table of Materials.
1. Preparation of stock solutions
NOTE: Prepare all stock solutions required for sample dilution at a PCR workstation to avoid contamination from foreign DNA.
<ol>
	<li>Prepare 10x PCR assay buffer, which consists of 500 mM of KCl, 100 mM of Tris-HCl, 15 mM of MgCl2, and 0.01 % (w/v) BSA. Measure the pH of the solution and adjust it to 8.3. 
	NOTE: This solution can be kept at room temperature for up to 1 month.</li>
	<li>Prepare the AAV dilution buffer, which consists of 1x PCR assay buffer, 0.2 ng/&#181;L salmon sperm sheared (sss) DNA, and 0.1 % pluronic F-68. Make this solution freshly with each assay and keep it at 4 &#176;C. 
	NOTE: If pluronic F-68 is not available, an alternative such as Poloxamer 188 non-ionic surfactant could be used.</li>
</ol>
2. Preparation of samples - DNase I treatment and serial dilution
NOTE: In order to remove DNA contaminants that could lower the accuracy of the vector genome titration, free DNA present within the AAV sample (including remaining plasmid DNA or non-encapsidated viral genomes) can be removed prior to PCR amplification using DNase. This is possible since the vector genomes are encapsidated within the AAV capsids and are therefore not accessible until the denaturing step of the PCR reaction itself (see step 5). Furthermore, since the vector genome number in any given sample is unknown, it is necessary to perform a serial dilution of the samples to ensure measurements remain within the upper and lower detectable limits. Perform all sample manipulations at a PCR workstation. Keep all samples on ice unless specified otherwise.
<ol>
	<li>Mix the AAV sample selected for analysis by briefly vortexing and then centrifuging to ensure all liquid remains at the bottom of the tube containing the sample.</li>
	<li>For DNase treatment, add 45 &#181;L of a solution containing 1x DNase I buffer, 0.1% pluronic F-68, and 0.04 U/&#181;L DNase I in DNase-free water into a 0.2 mL PCR 8-tube strip tube.</li>
	<li>Transfer 5 &#181;L of sample into the tube containing the DNAse solution. Vortex the samples and briefly centrifuge to ensure all liquid remains at the bottom.</li>
	<li>In a thermocycler, incubate the samples for 1 h at 37 &#176;C, then cool down to 4 &#176;C. Place on ice as soon as possible after 4 &#176;C is reached.</li>
	<li>Using a fresh 0.2 mL PCR 8-tube strip, make appropriate dilutions of the DNase-treated sample in AAV dilution buffer. Recommended sample dilutions depend on the expected vg titer (Table 2), but aim for at least 2 different dilutions per sample and run each dilution in duplicate. Ensure to rigorously (2,000-3,000 rpm) vortex the samples for 5 s before taking out any volume.</li>
	<li>After serial dilution, vortex the samples and centrifuge to ensure all liquid remains at the bottom of the tubes.</li>
	<li>Include a positive control of which vg titer is known, and a negative control (AAV dilution buffer only) which is also called the no template control (NTC). 
	NOTE: The positive control can be an in-house produced AAV or a commercial AAV reference material, which includes the target sequence of the primers/probe.</li>
</ol>
3. Preparation of dd_PCR master mix
NOTE: Select an appropriate target sequence within the vector genome and design a forward primer, reverse primer, and hydrolysis probes labeled with FAM or HEX reporter fluorophores following published guidelines13. Primers that target the transgene are preferred over primers that target the ITRs since the transgene is specific to the vector genome design, and the secondary structure associated with the hairpin formation of the ITRs can hamper efficient primer binding. Moreover, ITR primers might overestimate AAV titers if partially filled capsids that contain truncated vector genome fragments are present and still house either of the ITR sequences14. Prepare the dd_PCR master mix in a dedicated, separate workstation (pre-PCR room). This room should be separate from the one where the samples are prepared to avoid cross-contamination.
<ol>
	<li>Allow the primer, probe, and dd_PCR supermix reagents to reach room temperature (20-25 &#176;C). Afterward, vortex all reagents and briefly centrifuge the tubes to collect all liquid at the bottom of the tube.</li>
	<li>Prepare a volume of dd_PCR master mix in a separate microcentrifuge tube according to the required number of reactions by combining the forward and reverse primers, probe, dd_PCR supermix, and DNase-free water according to the volumes and concentrations described in Table 3.
	<ol>
		<li>Vortex the master mix and then briefly centrifuge the tube to ensure all liquid remains at the bottom of the tube. 
		NOTE: Always prepare extra dd_PCR master mix volume than required (n + 1) in order to account for pipetting volume loss.</li>
	</ol>
	</li>
	<li>Transfer 19.8 &#181;L of the master mix into each tube on a fresh 0.2 mL PCR 8-tube strip.</li>
</ol>
4. Droplet generation
NOTE: Samples and dd_PCR master mix are mixed separately prior to loading into a droplet-generating cartridge. Perform all manipulations at a PCR workstation, preferably a different one from where the samples were prepared, to avoid amplicon contamination. Alternatively, clean the PCR workstation thoroughly after preparing the samples.
<ol>
	<li>Transfer 2.2 &#181;L of the diluted sample into the 19.8 &#181;L volume of dd_PCR master mix in each tube of the PCR 8-tube strip and mix well. Vortex and briefly centrifuge to ensure all liquid remains at the bottom.</li>
	<li>Transfer 20 &#181;L of this solution into the wells contained in the middle &#34;sample&#34; row of a droplet-generating cartridge. Avoid bubbles in the droplet-generating cartridge; this can cause the instrument to give errors, and droplets will fail to be generated. 
	NOTE: Since the cartridges are in 8-well format, as are the PCR tube strips, it is recommended that an 8-channel pipette be used.</li>
	<li>Transfer 60 &#181;L of the droplet-generation oil to the wells contained on the lower &#34;oil&#34; row of the droplet-generating cartridge. 
	NOTE: Never leave a well of the droplet-generating cartridge empty; fill the empty sample well with dd_PCR buffer control for probes (with the corresponding volume described above).</li>
	<li>Place a rubber gasket over the droplet-generating cartridge and place it in the droplet generator.</li>
	<li>After the droplets are generated, use an 8-channel pipette to slowly transfer 42.5 &#181;L solution from the dd_PCR chip to a multi-well PCR plate. 
	NOTE: Pipette slowly and at a 45-degree angle to avoid droplet shearing in the liquid while pipetting. Visually inspect the plate so that all wells are filled with the same amount of liquid and droplets are visible as an opaque layer in the well.</li>
	<li>Seal the plate with an aluminum foil cover in a heat-sealing machine for 5 s at 180 &#176;C. 
	NOTE: Visually inspect that all wells are sealed. If not, repeat another 5 s at 180 &#176;C. The sealed plate can be kept for up to 4 h at 4 &#176;C before dd_PCR amplification in a thermal cycler.</li>
</ol>
5. dd_PCR amplification
NOTE: The thermal cycler should be placed in a separate room from the pre-PCR room to spatially separate pre-PCR activities from PCR amplification and avoid false-positive results due to amplicon contamination.
<ol>
	<li>Place the plate in a thermal cycler (with a 96-deep well reaction module) for PCR amplification and close it securely.</li>
	<li>Configure the thermocycler to run at the conditions described in Table 4 and at a reaction volume of 40 &#181;L. Include a pre-heating step in the program settings and ensure the lid is heated to 99 &#176;C.</li>
	<li>When the PCR program is over, keep the plate at 4 &#176;C for at least 15 min to ensure that the plate is fully cooled down. 
	NOTE: The plate can be kept for 48-72 h at 4 &#176;C before the droplets have to be read.</li>
</ol>
6. Droplet reading
<ol>
	<li>Load the plate into a droplet reader, input the following information in the system software, and continue the reading. 
	NOTE: Experiment type = Direct quantification; Supermix = dd_PCR supermix for probes (no dUTP); Assay type = single target per channel; Target info, signal channel 1 = FAM channel or HEX channel according to the used probe.</li>
	<li>Annotate the samples so that the position of each sample can be determined and identified effectively from the data file.</li>
</ol>
7. Data analysis
<ol>
	<li>Carry out the data analysis using the compatible software provided by the droplet reader manufacturer.</li>
	<li>Carry out a system suitability test to evaluate the overall performance (and, therefore, reliability) of the assay.
	<ol>
		<li>Check the event count (number of droplets) measured for each well on the PCR plate. Ideally, the event count is between 15,000 and 20,000. If the event count is &#60;10,000, then this value must be excluded from the analysis.</li>
		<li>Evaluate the 1D or 2D amplitude plot and check for the presence of droplet rain.</li>
		<li>Go to the data table tab and export the data to an Excel file.</li>
	</ol>
	</li>
	<li>Calculate vg titer following the steps below:
	<ol>
		<li>The working range of dd_PCR is 10-10 000 copies/&#181;L. Exclude any value that is &#60;10 copies/&#181;L from the analysis. The recommended range for the dd_PCR equipment used herein is 25-5000 copies/&#181;L. 
		NOTE: The data analysis software will provide a vector genome number in accordance with the PCR reaction volume (40 &#181;L), but this does not take into consideration the dilution factors applied during sample preparation.</li>
		<li>Calculate the sample vg titer per volume (typically expressed as vg/mL) by factoring in the various dilution factors throughout the sample preparation using the following relation: 
		NOTE: Concentration (value obtained from data analysis) x 10 (DNase I pre-treatment dilution) x 10 (PCR master mix dilution) x 1000 (&#181;L to mL) selected sample dilution (in AAV dilution buffer - e.g., Table 2).</li>
		<li>Calculate the coefficient of variation (%CV) between the replicates and between the different dilutions of the same sample. 
		NOTE: %CV is calculated by dividing the standard deviation by the mean. If the %CV between the different dilutions is &#62;20 %, the value should not be considered accurate.</li>
		<li>Check the values of the negative and positive control. The negative control should be less than 5 copies/&#181;L.</li>
	</ol>
	</li>
</ol>

Standardized Protocol for Viral Genome Quantification Using Digital Droplet PCR

<ol>
	<li>Balakrishnan, B., Jayandharan, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+biology+of+adeno-associated+virus+(AAV)+vectors+used+in+gene+therapy.">Basic biology of adeno-associated virus (AAV) vectors used in gene therapy.</a> Curr Gene Ther. 14 (2), 86-100 (2014).</li><li>Srivastava, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+tissue-tropism+of+adeno-associated+viral+vectors.">In vivo tissue-tropism of adeno-associated viral vectors.</a> Curr Opin Virol. 21, 75-80 (2016).</li><li>Pupo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV+vectors:+The+Rubik's+cube+of+human+gene+therapy.">AAV vectors: The Rubik's cube of human gene therapy.</a> Mol Ther. 30 (12), 3515-3541 (2022).</li><li>Gimpel, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+methods+for+process+and+product+characterization+of+recombinant+adeno-associated+virus-based+gene+therapies.">Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies.</a> Mol Ther Methods Clin Dev. 20, 740-754 (2021).</li><li>Brimble, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preventing+packaging+of+translatable+P5-associated+DNA+contaminants+in+recombinant+AAV+vector+preps.">Preventing packaging of translatable P5-associated DNA contaminants in recombinant AAV vector preps.</a> Mol Ther Methods Clin Dev. 24, 280-291 (2022).</li><li>Wright, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Product-related+impurities+in+clinical-grade+recombinant+AAV+vectors:+Characterization+and+risk+assessment.">Product-related impurities in clinical-grade recombinant AAV vectors: Characterization and risk assessment.</a> Biomedicines. 2 (1), 80-97 (2014).</li><li>Dobnik, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+quantification+and+characterization+of+adeno-associated+viral+vectors.">Accurate quantification and characterization of adeno-associated viral vectors.</a> Front Microbiol. 10, (2019).</li><li>Sidransky, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+ras+oncogene+mutations+in+the+stool+of+patients+with+curable+colorectal+tumors.">Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors.</a> Science. 256 (5053), 102-105 (1992).</li><li>Vogelstein, B., Kinzler, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+PCR.">Digital PCR.</a> Proc Natl Acad Sci USA. 96 (16), 9236-9241 (1999).</li><li>Hindson, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+droplet+digital+PCR+system+for+absolute+quantitation+of+DNA+copy+number.">High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.</a> Anal Chem. 83 (22), 8604-8610 (2011).</li><li>Pinheiro, L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+droplet+digital+polymerase+chain+reaction+format+for+DNA+copy+number+quantification.">Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification.</a> Anal Chem. 84 (2), 1003-1011 (2012).</li><li>Sanmiguel, J., Gao, G., Vandenberghe, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+digital+droplet-based+AAV+genome+titration.">Quantitative and digital droplet-based AAV genome titration.</a> Methods Mol Biol. 1950, 51-83 (2019).</li><li>. Planning droplet digital PCR experiments Available from: <a target="_blank" href="https://www.bio-rad.com/en-in/life-science/learning-center/introduction-to-digital-pcr/planning-ddpcr-experiments">https://www.bio-rad.com/en-in/life-science/learning-center/introduction-to-digital-pcr/planning-ddpcr-experiments</a> (2024)</li><li>Dorange, F., Le Bec, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+approaches+to+characterize+AAV+vector+production+&amp;+purification:+Advances+and+challenges.">Analytical approaches to characterize AAV vector production &amp; purification: Advances and challenges.</a> Cell Gene Ther Insights. 4 (2), 119-129 (2018).</li><li>Lock, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+determination+of+single-stranded+and+self-complementary+adeno-associated+viral+vector+genome+titers+by+droplet+digital+PCR.">Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR.</a> Hum Gene Ther Methods. 25 (2), 115-125 (2014).</li><li>Prantner, A., Maar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+concentration,+characterization,+and+integrity+analysis+of+recombinant+adeno-associated+viral+vectors+using+droplet+digital+PCR.">Genome concentration, characterization, and integrity analysis of recombinant adeno-associated viral vectors using droplet digital PCR.</a> PLoS One. 18 (1), e0280242 (2023).</li><li>Dobnik, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+quantification+and+characterization+of+adeno-associated+viral+vectors.">Accurate quantification and characterization of adeno-associated viral vectors.</a> Front Microbiol. 10, 1570 (2019).</li><li>Suoranta, T., Laham-Karam, N., Yla-Herttuala, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+protocol+for+accurate+titration+of+adeno-associated+virus+vectors.">Optimized protocol for accurate titration of adeno-associated virus vectors.</a> Hum Gene Ther. 32 (19-20), 1270-1279 (2021).</li><li>Droplet digital PCR applications guide. Bulletin 6407 Available from: <a target="_blank" href="https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf">https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf</a> (2024)</li></ol>

IVH, BM, and EH are co-founders of Tavira Therapeutics. The other authors declare no conflict of interest.

Accurate quantification of AAV vector genome copies in vector preparations is essential for the development of AAV-based gene therapy products. Several methods exist for determining the vg titer, with quantitative PCR (qPCR) and dd_PCR being the two most widely used and accepted techniques. dd_PCR is often preferred over qPCR due to its independence from amplification efficiency, higher precision, and greater robustness15. Numerous protocols for vector genome titration by dd_PCR are available in the literature, each with its own sample preparation methods15,16,17. However, there is a lack of a qualified consensus protocol. This article presents a validated fit-for-purpose dd_PCR protocol for the quantification of AAV vector genomes in purified vector samples (Figure 2).
Close attention to sample handling is critical when performing this protocol. Cross-contamination can pose a significant challenge in accurately assessing vg titers; therefore, samples are best processed under a PCR workstation to avoid contamination with foreign DNA. Additionally, proper execution of DNase incubation is crucial to eliminate unpackaged contaminant DNA without disrupting the capsid and potentially digesting the packaged vector genome. Many protocols include heat inactivation and proteinase K treatment16,17,18. However, during in-house method development, it was found that excessive heating was detrimental to vg titration, and proteinase K treatment was unnecessary (data not shown).
As dd_PCR becomes increasingly popular for quantifying viral genomes, manufacturers have published detailed application guides with guidelines on designing and optimizing dd_PCR assays19. Troubleshooting information is also available. In practice, when an assay is correctly designed, the most frequent issues include droplet rain and low droplet count. Droplet rain is often caused by poor accessibility of the primer/probes to the amplicon, but several strategies can address this issue. For instance, a temperature gradient experiment can help establish the optimal annealing temperature. Additionally, diluting samples to lower the DNA amount (recommended to be less than 66 ng) or performing a restriction digest with specific enzymes that cut outside the amplicon region can improve template accessibility. A recommendation of 10 U of restriction enzyme per &#181;g of DNA is typically effective. The issue of low droplet count (fewer than 10,000) is frequently due to poor pipetting of the sample and oil in the cartridges. Slow pipetting with appropriate tips is recommended to avoid shearing of the droplets. While the dd_PCR assay is robust, one limitation is the extended time to result. The process from sample treatment to droplet reading takes several hours, which can be a drawback when results are needed quickly.
Accurate quantification of AAV genome copies in vector preparations is necessary throughout all stages of the AAV gene therapy life cycle. Specifically, it is important for optimizing production and purification processes, conducting preclinical studies, and determining clinical dosages for AAV-based gene therapy products. The dd_PCR protocol presented here is broadly applicable and can be used on purified AAV products with different serotypes and transgenes.

Gene therapy is a therapeutic modality commonly used to treat genetic disorders. The design of any given gene therapy is specific to the associated pathology of the target indication, but all gene therapies involve the intracellular delivery of genetic material to target cells in order to elicit a therapeutic effect1. Gene therapy can be further classified into several categories, including gene replacement for loss-of-function mutations, gene silencing for gain-of-function abnormalities, and gene editing techniques. Irrespective of the specific strategy employed, the therapeutic nucleic acid material (referred to as a transgene) must be packaged within a vector in order to achieve targeted intracellular delivery2.
Although a variety of viral and non-viral vector systems are available for gene therapy development, adeno-associated viruses (AAVs) are frequently chosen due to the broad viral tropism and low immunogenicity associated with this group of viruses1,2. To date, seven gene therapies utilizing AAV for therapeutic gene delivery have achieved approval from either the European Medicines Agency (EMA) or the Food and Drug Administration (FDA) targeting diseases ranging from hemophilia (e.g., Roctavian) to spinal muscular atrophy (e.g., Zolgensma)3.
Production of AAV-based gene therapies stems from an understanding of the wild-type AAV itself. AAV is a small DNA virus within the Parvoviridae family comprising 13 main serotypes (AAV1-13)3. The AAV genome comprises a ~4.7 kb, single-stranded DNA molecule containing two main open reading frames (ORFs) that encode the essential viral genes necessary for genome replication, capsid assembly, and packaging (rep, cap). The viral genome is flanked at both the 5&#39; and 3&#39; ends by palindromic nucleotide sequences, referred to as inverted terminal repeats (ITRs). These ITRs form hairpin-like structures that play crucial roles in genome replication and packaging of de novo viral genomes into newly synthesized viral capsids. AAV is a helper-dependent virus and, therefore, requires the expression of ancillary genes from other viruses, such as herpes simplex virus (HSV) or adenovirus (AdV) in order to become replication-competent1.
In order to produce AAVs, a suitable cell-based expression system is employed to facilitate the expression of the viral capsid proteins and subsequent assembly into de novo viral particles, followed by encapsidation of an ITR-flanked selected transgene (also referred to as a vector genome). This process commonly utilizes a triple plasmid system, comprising (1) a plasmid harboring helper genes derived from a helper virus, (2) a plasmid encoding the essential viral elements (rep/cap), and (3) a plasmid carrying the therapeutic expression cassette (commonly referred to as a transfer plasmid)4. The unique presence of inverted terminal repeat (ITR) packaging signals flanking the therapeutic expression cassette in the transfer plasmid ensures specific packaging of the transgene, while mostly excluding the viral genes present on the other plasmids. Co-transfection of this three-plasmid system into a cell-based expression platform (typically HEK293T cells) results in the production of transduction-competent, replication-deficient viral particles suitable for use in gene therapy applications3,4.
There are a number of critical quality attributes (CQAs) associated with the production of AAV-based gene therapies that must be assessed in order to ensure the potency, purity, and safety of the intended medicinal product4. These CQAs include virus titer, capsid content, and aggregation. Virus titer itself is a combination of the number of viral particles (capsid titer) and the number of vector genomes (vector genome titer) present in any given preparation. Ideally, the ratio between these two titers should be 1:1 since each viral particle should contain one vector genome, but inefficiencies in vector genome packaging during biosynthesis result in the co-production of empty or partially filled capsids (those containing partial vector genome sequences and/or non-vector genome sequences)5. The presence of such impurities can potentially evoke unwarranted immune responses and compete for vector-binding sites, thereby increasing the risk of immunotoxicity and reducing the rate of transduction of full capsids6. Accurate quantification of AAV genomes is therefore essential for establishing virus titer and capsid content. This impacts both basic research as well as the gene therapy industry, which requires accurate dosage in order to maintain both the safety and efficacy of medicinal products.
Digital Droplet polymerase chain reaction (dd_PCR) has become closely associated with the quantification of virus titer since it can be utilized to determine the number of vector genomes present in any given preparation7. Digital PCR itself was first introduced in the 1990s8,9&#160;and dd_PCR is an enhancement to this technology that allows for high-throughput sample processing10,11. In dd_PCR, a 20 &#181;L real-time PCR reaction is partitioned into approximately 20,000 oil-encased droplets, giving up to 96 such reactions when accommodated on a standard 96-well plate. Compared with conventional quantitative PCR (qPCR), dd_PCR offers several advantages, including enhanced sensitivity, increased precision, and more direct and absolute quantification of target sequences without the need for standard curves. Moreover, the high level of partitioning in dd_PCR reduces the impact of PCR inhibitors and minimizes the potential for bias from preferential amplification of certain templates, making it an attractive option for analytical measurement of vector genome titration.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>8-channel pipette 10 &#181;L</td>
			<td>Eppendorf</td>
			<td>3,12,50,00,010</td>
			<td></td>
		</tr>
		<tr>
			<td>8-channel pipette 200 &#181;L</td>
			<td>Eppendorf</td>
			<td>3,12,50,00,036</td>
			<td></td>
		</tr>
		<tr>
			<td>8-channel pipette 300 &#181;L</td>
			<td>Eppendorf</td>
			<td>3,12,50,00,052</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well PCR strip</td>
			<td>Sarstedt</td>
			<td>72.991.002</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>C1000 Touch Thermal Cycler with 96&#8211;Deep Well Reaction Module</td>
			<td>Bio-Rad</td>
			<td>1851197</td>
			<td></td>
		</tr>
		<tr>
			<td>ddPCR Buffer Control for Probes 9 mL (2 x 4.5 mL)</td>
			<td>Bio-Rad</td>
			<td>1863052</td>
			<td></td>
		</tr>
		<tr>
			<td>ddPCR Supermix for Probes (No dUTP) (2 x 1 mL)</td>
			<td>Bio-Rad</td>
			<td>1863023</td>
			<td></td>
		</tr>
		<tr>
			<td>ddPCR 96-Well Plates (pkg of 25)</td>
			<td>Bio-Rad</td>
			<td>12001925</td>
			<td></td>
		</tr>
		<tr>
			<td>ddPCR Droplet Reader Oil (2 x 1L)</td>
			<td>Bio-Rad</td>
			<td>1863004</td>
			<td></td>
		</tr>
		<tr>
			<td>DG8 Cartridge Holder</td>
			<td>Bio-Rad</td>
			<td>1863051</td>
			<td></td>
		</tr>
		<tr>
			<td>DG8 Cartridges for QX200/QX100 (pkg of 24)</td>
			<td>Bio-Rad</td>
			<td>1864008</td>
			<td></td>
		</tr>
		<tr>
			<td>DG8 Gaskets for QX200/QX100 (pkg of 24)</td>
			<td>Bio-Rad</td>
			<td>1863009</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I (10U/&#181;L) + buffer</td>
			<td>Roche</td>
			<td>4716728001</td>
			<td></td>
		</tr>
		<tr>
			<td>Droplet Generation Oil for Probes (10 x 7 mL)</td>
			<td>Bio-Rad</td>
			<td>1863005</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf ep Dualfilter T.I.P.S. Filter Tip, 0.1-10 &#956;L, 34 mm, Rack, PCR Clean, STERILE&#160;</td>
			<td>Eppendorf</td>
			<td>30078500</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf ep Dualfilter T.I.P.S. Filter Tip, 20-300 &#956;L, 55 mm, Rack, PCR Clean, STERILE&#160;</td>
			<td>Eppendorf</td>
			<td>30078560</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf ep Dualfilter T.I.P.S. Filter Tip, 2-100 &#956;L, 53 mm, Rack, PCR Clean, STERILE&#160;</td>
			<td>Eppendorf</td>
			<td>30078543</td>
			<td></td>
		</tr>
		<tr>
			<td>Forward lyophilized primers and respective master stocks at 100 mM&#160;</td>
			<td>IDT</td>
			<td></td>
			<td>GFP as target sequence. Forward primer: 5&#39;-GAACGGCATCAAGGTGAACT-3&#39;</td>
		</tr>
		<tr>
			<td>Lyopohilized probe and master stock at 100 &#181;M&#160;</td>
			<td>IDT</td>
			<td></td>
			<td>GFP as target sequence. PrimeTime Eco Probe: /56-FAM/CAAGATCCG/ZEN/CCACAACATCGAGGA/3IABkFQ/</td>
		</tr>
		<tr>
			<td>Magnesium Chloride (MgCl2)</td>
			<td>Chemlab Analytical</td>
			<td>CL00.1381</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease free water</td>
			<td>IDT</td>
			<td>11-04-02-01</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Plate Heat Seal, foil, pierceable (pkg of 100)</td>
			<td>Bio-Rad</td>
			<td>1814040</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-68 non-ionic surfactant (100x)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>24040032</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Honeywell research chemicals</td>
			<td>31248</td>
			<td></td>
		</tr>
		<tr>
			<td>QX manager software</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>Software to analyse ddPCR data</td>
		</tr>
		<tr>
			<td>QX200 Droplet Generator</td>
			<td>Bio-Rad</td>
			<td>1864002</td>
			<td></td>
		</tr>
		<tr>
			<td>QX200 Droplet Reader</td>
			<td>Bio-Rad</td>
			<td>1864003</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent reservoir</td>
			<td>VWR</td>
			<td>613-1181</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse lyophilized primers and respective master stocks at 100 mM&#160;</td>
			<td>IDT</td>
			<td></td>
			<td>GFP as target sequence. Reverse primer: 5&#39;-TGCTCAGGTAGTGGTTGTCG-3&#39;</td>
		</tr>
		<tr>
			<td>SafeSeal reaction tube, 1.5 mL</td>
			<td>Sarstedt</td>
			<td>72.706.200</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmon Sperm DNA, sheared (10 mg/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9680</td>
			<td></td>
		</tr>
		<tr>
			<td>TE buffer</td>
			<td>IDT</td>
			<td></td>
			<td>Accompanied by primers when ordering</td>
		</tr>
		<tr>
			<td>Tris hydrochloride (Tris-HCl)</td>
			<td>Roche</td>
			<td>10812846001</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of adeno-associated viral genomes in purified vector samples by digital droplet polymerase chain reaction

Adeno-associated virus (AAV) is a non-pathogenic virus used as a delivery vehicle to transfer therapeutic genes into patients. Accurate quantification of AAV genome copy number in vector preparations is essential for bioprocess optimization and dosage calculation in both preclinical and clinical studies of AAV-based gene therapy products. Currently, a consensus protocol for AAV viral genome titration is lacking. Here, we present a digital droplet PCR (dd_PCR) protocol for the quantification of viral genomes in purified vector samples. Samples are treated with DNase I to eliminate unpackaged contaminant DNA. DNase-treated samples are then mixed with an appropriate primer-probe set (designed according to the target AAV genome) and PCR reagents, and then loaded into a droplet generator. The prepared droplets are transferred into a PCR plate, where PCR amplification is performed and analyzed. The viral genome titer is calculated based on the concentration (copies/&#181;L), accounting for sample dilutions. A successful measurement shows a clear separation of the positive and negative droplet clouds, has at least 10,000 accepted droplets, shows a value between 10 copies/&#181;L and 10,000 copies/&#181;L, and has a coefficient of variation (CV) between repeats lower than 20%. Reliable viral genome titration will aid in the development of safe and effective AAV-based gene therapy products.

Results can be visualized using appropriate software. For each well, the 1D amplitude plot displays all droplets relative to their amplitude. A clear separation between positive and negative droplets is expected. If more than 10% of the total droplets are found between the positive and negative droplet clouds (a phenomenon referred to as droplet rain), it is necessary to re-measure the sample (see Figure 1). Additional information on droplet rain is available in the discussion.
A data table can be created to summarize all recorded information, including the sample name, number of accepted droplets (event counts), and concentration (copies/&#181;L). Ideally, the event count should fall between 15,000 and 20,000 accepted droplets. If the event count for a well is below 10,000, the data point should be excluded from the analysis. An example of the output data is provided in Table 5.
The vg titer can be calculated based on the concentration (copies/&#181;L), with consideration of sample dilutions. The working range of dd_PCR is 10-10,000 copies/&#181;L. Values below 10 copies/&#181;L should be excluded from the analysis. The vg titer for samples, as well as the positive and negative controls, should be calculated. The measured value of the positive control should have a coefficient of variation (%CV) lower than 20% relative to the theoretical value. The negative control should be less than 5 copies/&#181;L. Additionally, the %CV between repeats and different dilutions for each sample should be calculated. If the %CV between different dilutions exceeds 20%, the value may be considered inaccurate, and the sample may need to be re-measured.
A successful measurement is characterized by a clear separation of the positive and negative droplet clouds, at least 10,000 accepted droplets, a value between 10 copies/&#181;L and 10,000 copies/&#181;L, and a %CV between repeats lower than 20%.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67252/67252fig01.jpg" /> 
Figure 1: Visualization of dd_PCR droplets. (A) The 1D amplitude plot demonstrates a clear separation of positive and negative droplets, indicating successful droplet partitioning. (B) The plot shows a poor separation of positive and negative droplets, known as droplet rain, which suggests suboptimal partitioning or potential issues with the assay. <a href="https://www.jove.com/files/ftp_upload/67252/67252fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67252/67252fig02.jpg" /> 
Figure 2: Workflow for quantifying viral genomes using digital droplet PCR. (1) Buffers, reagents, and solutions are prepared according to the manufacturer&#39;s instructions or step 1 of the protocol (Preparation of stock solutions). (2) 45 &#181;L of the DNase I solution is aliquoted into each tube of an 8-tube PCR strip. After vortexing and briefly centrifuging the samples, 5 &#181;L of the sample is added to one of the tubes. The DNase I and sample-containing eight-tube PCR strip are incubated in a thermocycler for 1 h at 37 &#176;C, followed by serial dilutions of the samples. (3) The PCR mastermix is prepared as described, and 19.8 &#181;L is aliquoted into each tube of an 8-tube PCR strip. Sample serial dilutions from step 2 are added to the mastermix. 20 &#181;L of the mastermix plus 
sample solution is loaded into the middle row&#160;of the cartridge, and 60 &#181;L of droplet-generation oil is transferred to the lower wells&#160;of a droplet-generating cartridge.&#160;The cartridge is then placed into the droplet generator and run according to specified conditions. After droplet generation, 42.5 &#181;L from the top row of the cartridge is transferred to a multi-well PCR plate. (4) The PCR plate is loaded into a PCR thermocycler and run according to the provided conditions. The plate is analyzed using a dd_PCR reader. <a href="https://www.jove.com/files/ftp_upload/67252/67252fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Precision data. This table presents precision data from four sets of quality control (QC) samples, each with five concentrations (QC1-QC5). Each QC was measured five times. The coefficient of variation (%CV) between different runs, representing repeatability (A), and between different sets of samples, representing intermediate precision (B), is shown. <a href="https://www.jove.com/files/ftp_upload/67252/67252_Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Sample dilutions. The table shows the recommended sample dilutions based on the expected viral genome titer (vg/mL). A total of three dilutions are recommended to ensure at least two values fall within the working range of the assay. <a href="https://www.jove.com/files/ftp_upload/67252/67252_Table 2.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: dd_PCR master mix composition. This table outlines the composition of the dd_PCR master mix, which includes a forward and reverse primer (909 nM), probe (227 nM), and dd_PCR supermix for probes (without dUTP, 1x). <a href="https://www.jove.com/files/ftp_upload/67252/67252_Table 3.xlsx" target="_blank">Please click here to download this Table.</a>
Table 4: Thermocycling conditions. The table details the recommended PCR program, which includes: (1) a 10 min incubation at 95 &#176;C for capsid disruption and enzyme activation, (2) 40 cycles of 30 s at 94 &#176;C for DNA denaturation, and 1 min at 60 &#176;C for annealing and extension, (3) a 10 min incubation at 98 &#176;C for enzyme deactivation, and (4) holding at 4 &#176;C. The annealing temperature may require optimization based on the primer/probe set used. <a href="https://www.jove.com/files/ftp_upload/67252/67252_Table 4.xlsx" target="_blank">Please click here to download this Table.</a>
Table 5: Example of output data from an actual AAV run. The table provides an example of output data from an actual AAV run, where one sample was measured in two different dilutions, each in duplicate. The viral genome (vg) titer (in vg/mL) is calculated using the following formula: 10 (DNase I pre-treatment) x 10 (dilution in the master mix) x 1,000 (&#181;L to mL) x dilution in AAV dilution buffer. STDEV represents standard deviation, and CV indicates the coefficient of variation. <a href="https://www.jove.com/files/ftp_upload/67252/67252_Table 5.xlsx" target="_blank">Please click here to download this Table.</a>

Author Spotlight: Optimizing Digital Droplet PCR Method for Accurate Adeno-Associated Viral Genome Quantification

Precise quantification of adeno-associated virus (AAV) vector genome copies is critical, but a standardized protocol has yet to be established. This protocol describes a validated method for preparing purified AAV samples and conducting digital droplet polymerase chain reaction (dd_PCR) to reliably quantify the viral genome titer.

Quantification of Adeno-Associated Viral Genomes in Purified Vector Samples by Digital Droplet Polymerase Chain Reaction

Adeno-associated virus (AAV) is a non-pathogenic virus used as a delivery vehicle to transfer therapeutic genes into patients. Accurate quantification of ...

quantification-adeno-associated-viral-genomes-purified-vector-samples

Research

JoVE Journal

Bioengineering

1.6K Views.  KU Leuven. Precise quantification of adeno-associated virus (AAV) vector genome copies is critical, but a standardized protocol has yet to be established. This protocol describes a validated method for preparing purified AAV samples and conducting digital droplet polymerase chain reaction (dd_PCR) to reliably quantify the viral genome titer.

Video: Author Spotlight: Optimizing Digital Droplet PCR Method for Accurate Adeno-Associated Viral Genome Quantification

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

Enhanced darkfield microscopy and hyperspectral imaging with spectral mapping enable screening, localization, and identification of nanoscale materials in histological samples with improved speed and accuracy over traditional methods. The goal of this paper is to provide methods for darkfield imaging and hyperspectral mapping of metal oxide nanoparticles in histological samples.

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different bone loss conditions. However, the standard approach for trabecular analysis of micro-CT images is slice-by-slice semi-automatic hand-contouring, which is labor intensive and error prone. Described here is an efficient method for automatic segmentation of trabecular bones according to the bone's outer boundaries, where trabecular bones can be identified and segmented automatically with accuracy with less operator bias when appropriate segmentation parameters are set. To profile satisfactory segmentation parameters, an image stack of segmentation results is displayed, where all possible combinations of the segmentation parameters are changed one by one in sequence, and segmentation results with associated parameters can easily be visually checked. As a quality-control feature of the plugin, simulated standard objects are quantified where the measured quantities can be compared with theoretical values. Layer-by-layer quantification of trabecular properties and trabecular thicknesses are reported by such a plugin, and the distributions of such properties within the selected regions can be profiled easily. Although layer-by-layer quantification retains more information about trabecular bones and facilitates further statistical analysis of structural changes, such measures are unavailable from the output of current commercial software, where only a single quantified value for each parameter is reported for each sample. Therefore, the described workflows are better approaches for analyzing trabecular bones with accuracy and efficiency.

We present a workflow for segmenting and quantifying trabecular bones for 2D and 3D images based on the bone&#39;s outer boundary using an ImageJ plugin. This approach is more efficient and accurate than the current manual hand-contouring approach, and provides layer-by-layer quantifications, which are not available in current commercial software.

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different ...

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and morphology of the obtained microsphere. A microsphere copolymerized with an acrydite-DNA probe was successfully fabricated. Different methods such as asymmetric PCR, exonuclease digestion, and isolation on streptavidin-coated magnetic beads can be used to synthesize single-stranded DNA (ssDNA). However, these methods cannot efficiently use large amounts of highly purified ssDNA. Here, we describe a microsphere-PCR protocol detailing how ssDNA can be efficiently amplified and separated from dsDNA simply by pipetting from a PCR reaction tube. The amplification of ssDNA can be applied as potential reagents for the DNA microarray and DNA-SELEX (Systematic evolution of ligands by exponential enrichment) processes.

This work provides a method for the fabrication of droplet-based microfluidic platforms and the application of polyacrylamide microspheres for microsphere-PCR amplification. The microsphere-PCR method makes it possible to obtain single-stranded DNA amplicons without separating double-stranded DNA.

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Bulk Droplet Vitrification for Primary Hepatocyte Preservation

Vitrification is a promising ice-free alternative for classic slow-freezing (at approximately 1 &#176;C/min) cryopreservation of biological samples. Vitrification requires extremely fast cooling rates to achieve transition of water into the glass phase while avoiding injurious ice formation. Although pre-incubation with cryoprotective agents (CPA) can reduce the critical cooling rate of biological samples, high concentrations are generally needed to enable ice-free cryopreservation by vitrification. As a result, vitrification is hampered by CPA toxicity and restricted to small samples that can be cooled fast. It was recently demonstrated that these inherent limitations can be overcome by bulk droplet vitrification. Using this novel method, cells are first pre-incubated with a low intracellular CPA concentration. Leveraging rapid osmotic dehydration, the intracellular CPA is concentrated directly ahead of vitrification, without the need to fully equilibrate toxic CPA concentrations. The cellular dehydration is performed in a fluidic device and integrated with continuous high throughput generation of large sized droplets that are vitrified in liquid nitrogen. This ice-free cryopreservation method with minimal CPA toxicity is suitable for large cell quantities and results in increased hepatocyte viability and metabolic function as compared to classical slow-freezing cryopreservation. This manuscript describes the methods for successful bulk droplet vitrification in detail.

This manuscript describes an ice-free cryopreservation method for large quantities of rat hepatocytes whereby primary cells are pre-incubated with cryoprotective agents at a low concentration and vitrified in large droplets.

Vitrification is a promising ice-free alternative for classic slow-freezing (at approximately 1 &#176;C/min) cryopreservation of biological samples. ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...