The authors acknowledge Liyi Xu for providing MDA reagents and protocols. We also thank David Feldman and Navpreet Ranu for discussions relating to this work. This work was supported in part by a Burroughs Wellcome Career Award at the Scientific Interface to PCB.

Note: Fabricating the microfluidic device is not necessary for this assay, as droplets for this protocol can be formed with existing commercial droplet makers23,29.
1. Make the Droplet-forming Microfluidic Device
<ol>
	<li>Prepare master mold for the channels with the SU-8 Master Fabrication protocol outlined previously31, but with a droplet generator mask pattern32.</li>
	<li>Fabricate devices in PDMS using the techniques of soft lithography32,33.</li>
</ol>
2. Prepare Reaction Mix for Bulk Droplet Readout
 
Note: The protocol can be used to quantify nucleic acids using many types of amplification reactions. As an example, the reagents needed for MDA of several Lambda DNA concentrations are given. Reagent details are listed in the Table of Specific Reagents. The distribution of template within the droplets depends on sufficient mixing of the sample.
<ol>
	<li>Obtain or Purify Phi29 DNA polymerase.
	<ol>
		<li>Purify Phi29 as previously described7, or purchase from supplier. The Phi29 used for the shown experiments was purified.</li>
	</ol>
	</li>
	<li>Prepare 10 ml&#160;of Denaturation Buffer consisting of 65 mM KOH, 1.65 mM EDTA, and 14 mM DTT.</li>
	<li>Prepare 10 ml&#160;of Neutralization Buffer consisting of 65 mM HCl, 0.21 M Tris-Cl pH 7.0, and 9 mM Tris-Cl pH 8.0.</li>
	<li>Prepare two standards, one no template control (NTC) such as nuclease-free water, and one high template concentration (around 4 pg/&#181;l&#160;Lambda DNA). Denature 3.3 &#181;l&#160;of each standard with 3.3 &#181;l&#160;of Denaturation Buffer in a qPCR tube. Incubate at RT for 3 min. Quench each with 3.3 &#181;L of Neutralization Buffer.</li>
	<li>Denature 3.3 &#181;l&#160;of the template of interest with 3.3 &#181;l&#160;of Denaturation Buffer in a qPCR tube. Incubate at RT for 3 min. Quench with 3.3 &#181;l&#160;of Neutralization Buffer.</li>
	<li>Prepare 11 &#181;l&#160;of master mixture per sample on ice. The 2x MDA reaction master mixture consists of 2x double-stranded DNA (dsDNA) binding dye, 2x qPCR reference dye, 50 &#181;M randomized oligo, 2 mg/ml&#160;BSA, 2x Phi29 DNA Polymerase Reaction Buffer, 4.8 mM dNTPs, nuclease-free water, and 40 &#181;g/ml&#160;Phi29 DNA polymerase. Add Phi29 DNA polymerase last to ensure the polymerase does not encounter pH levels much higher or lower than 7.5.Mix well.</li>
	<li>Optional: For fewer false positives, expose master mix with UV light prior to forming droplets34.
	<ol>
		<li>Prepare 10 &#181;l&#160;of pre-mixture per sample in a 0.5 ml&#160;or 1.5 ml&#160;clear tube on ice. The pre-mix consists of 55 &#181;M randomized oligo, 2.2 mg/ml&#160;BSA, 1.1x Phi29 DNA Polymerase Reaction Buffer, 5.3 mM dNTPs, and nuclease-free water.</li>
		<li>Place the tube in water on ice as described34 and expose to UV light (254 nm) to an accumulated dose of 5.7 J/cm2.</li>
		<li>Add dsDNA-binding dye to 1x, reference dye to 1x, and Phi29 to 40 &#181;g/ml&#160;in the pre-mixture. Mix well.</li>
	</ol>
	</li>
	<li>Combine 10 &#181;l&#160;of denatured template or standard and 10 &#181;l&#160;of master mixture. Mix well.</li>
</ol>
3. Droplet Formation
Note: Droplets can be very sensitive to static electricity and shear stress. Remove clothes that may cause static electricity, and ground yourself before forming droplets. Handle tubes of emulsion from the top of the tube, as far from the emulsion as possible. Pipette emulsions very slowly, preferably with a wide bore pipet tip. When dispensing from a pipette tip, watch the emulsion on the side of the tip to determine pipetting speed.
<ol>
	<li>Form droplets. For the experiments shown, the microfluidic device has an oil inlet and two aqueous inlets with a flow-focusing junction for generating droplets. Use two syringe pumps to control the flow rates of 55 &#181;l&#160;of oil with surfactant and a 20 &#181;l&#160;of reaction mixture through the microfluidic chip to generate 20,000 1 nl droplets.</li>
	<li>Note: It is possible to produce droplets with many types of oil with surfactant, but the composition will greatly affect droplet stability at high temperatures and over time. One possible combination is fluorinated oil HFE 7500 with an anionic surfactant19,35.</li>
	<li>Produce droplets at any size with any method13,36, but the size variability in the droplet population will affect the bulk fluorescence, and therefore the accuracy of the assay. For the experiments shown, droplets were monodisperse with a volume of 1 nl.</li>
	<li>Collect all of the droplets and oil into PCR tubes. For each sample, aliquot 30 &#181;l&#160;of oil into fresh PCR tubes with optical caps, and transfer 20 &#181;l&#160;of droplets on top of the oil. The consistent volumes ensure the assay is as accurate as possible.</li>
	<li>Close tube caps tightly, as loose caps will allow the oil to evaporate.</li>
</ol>
4. Isothermal Amplification
<ol>
	<li>Using a PCR thermocycler, qPCR thermocycler, or hot plate, incubate the samples at 30 &#176;C for 7 hr, and inactivate for 1 min at 75 &#176;C. The tubes of droplets may be left in a fridge covered with foil for a few hours at this point.</li>
</ol>
5. Data Acquisition and Analysis
<ol>
	<li>Immediately following reaction inactivation, measure dye fluorescence levels for all samples using the qPCR thermocycler. Optimal filter settings will depend on dye excitation and emission spectra. For this example, use the 492 nm-516 nm filter set for the dsDNA-binding dye, and the 585 nm-610 nm filter set for the reference dye. Other fluorescent measurement techniques may be used to quantify the fluorescence.</li>
	<li>For each sample, divide the fluorescence intensity of the dsDNA-binding dye by the fluorescence intensity of the reference dye. Subtract the background fluorescence, or the normalized fluorescence of the no template control, from all samples.</li>
	<li>Create a linear standard curve using the corrected fluorescence measurements from the standards. The NTC standard represents the fluorescence for a sample with 0% fluorescent droplets (&#8220;all negative&#8221;), and the high template concentration fluorescence measurement is the expected fluorescence for a sample with 100% fluorescent droplets (&#8220;all positive&#8221;). Using the corresponding standard curve, predict the intermediate ratios of positive and negative droplets based on their bulk fluorescence.</li>
</ol>
6. Producing Droplets for Proof-of-Principle Measurements
<ol>
	<li>Make fluorescent (positive) droplets: Prepare a PCR reaction mixture consisting of 0.1 ng Lambda DNA, 1x dsDNA-binding dye, 1x reference dye, 1.5 units Taq DNA polymerase, 10 mM Tris-HCl, 50mM KCl, 1.5 mM MgCl2, 0.2 mM dNTP, and 0.5 &#181;M primers. Thermocycle the mixture 30 times (95 &#176;C for 20 sec, 55 &#176;C for 20 sec, 72 &#176;C for 1 min), then form droplets as described above.</li>
	<li>Make non-fluorescent (negative) droplets: Prepare a PCR reaction mixture consisting of 1x dsDNA-binding dye, 1x reference dye, 1.5 units Taq DNA polymerase, 10 mM Tris-HCl, 50mM KCl, 1.5 mM MgCl2, 0.2 mM dNTP, and 0.5 &#181;M primers. Thermocycle the mixture 30 times (95 &#176;C for 20 sec, 55 &#176;C for 20 sec, 72 &#176;C for 1 min), then form droplets.</li>
	<li>Forming pre-mixed droplet ratios
	<ol>
		<li>Distribute 20 &#181;l&#160;of fluorinated oil into qPCR tubes, cover with an optical cap to prevent evaporation.</li>
		<li>Gently pipette 10 &#181;l&#160;of well-mixed droplets in desired positive:negative ratios on top of the oil. In the results shown, positive:negative droplet ratios were 1 (all positive), 0.8, 0.5, 0.2, 0.1, 0.01, 0.001, 0.0001, and 0 (all negative).</li>
	</ol>
	</li>
	<li>Measure dye fluorescence levels for all samples using the qPCR thermocycler. For this protocol, use the 492 nm-516 nm filter set for the dsDNA-binding dye, and the 585 nm-610 nm filter set for the reference dye.
	<ol>
		<li>Normalize dsDNA binding dye fluorescence using reference dye fluorescence. Further normalize by the fluorescence of the &#8220;all positive&#8221; sample.</li>
	</ol>
	</li>
	<li>Create a linear standard curve using the normalized fluorescence measurements from the &#8220;all positive&#8221; and &#8220;all negative&#8221; samples. Using the corresponding standard curve, predict the intermediate ratios of positive and negative droplets based on their bulk fluorescence.</li>
	<li>Repeat the experiment for each ratio (n = 3) with independent droplet formation and mixing for each replicate.</li>
</ol>
7. Proof-of-Principle MDA Experiment
<ol>
	<li>Measure the concentration of Lambda DNA stock solution with a spectrophotometer.
	<ol>
		<li>Calculate the template concentration necessary to average 10 template molecules per droplet. For this protocol, assume droplet volumes are 1 nl&#160;and 1 ng of Lambda DNA contains approximately 1.9 x 107 copies. Therefore, 10 templates per droplet will be 10 copies per nl, or 526 fg Lambda DNA per &#181;l. The template will be diluted ~6x when droplets are formed, therefore the initial highest template concentration will be 3.2 pg/&#181;l.</li>
	</ol>
	</li>
	<li>Serially dilute the Lambda DNA to 3.2 pg/&#181;l&#160;with Denaturation Buffer and equal volume Neutralization Buffer. For a dilution factor of 0.1, combine 10 &#181;l&#160;sample with 45 &#181;l&#160;of Denaturation Buffer and incubate at RT for 3 min. Add 45 lL of Neutralization Buffer to quench.</li>
	<li>Further dilute the sample as described in step 7.2 to make the rest of the samples for the experiment. The initial template concentrations in the reactions shown were 3.2 pg, 320 fg, 160 fg, 32 fg, 16 fg, 3.2 fg, and 1.6 fg per microliter. 
	Note: The final template concentrations after mastermix addition were 526 fg, 52.6 fg, 26.3 fg, 5.26 fg, 2.63 fg, 526 ag, and 263 ag per microliter. 10, 1, 0.5, 0.1, 0.05, 0.01, and 0.005 expected Lambda copies per droplet, respectively.</li>
	<li>Generate and analyze droplets from each sample as described in steps 2.5-5.3.</li>
	<li>Acquire the fluorescence images shown (Figure 2,3) using a digital camera mounted to an epi-fluorescence microscope with a 10x objective at RT. Acquire twelve fields of view for each sample. Correct fluorescence images by a background image obtained with a fluorescent slide.</li>
	<li>Use a contained chamber for imaging37 as the emulsion oil will evaporate quickly.</li>
	<li>Analyze individual droplet intensities using an ImageJ macro (Supplemental code file).
	<ol>
		<li>Choose a threshold fluorescence intensity that best separates non-fluorescent droplets in the NTC images from the fluorescent droplets in the high template concentration images. For each unknown sample, calculate the fraction of droplets with a fluorescence intensity above the threshold.</li>
	</ol>
	</li>
</ol>
8. PCR and MDA bulk reactions
<ol>
	<li>Prepare PCR reaction.
	<ol>
		<li>Serially dilute template DNA with a dilution factor of 0.1. For each dilution, combine 10 &#181;l&#160;of template with 45 &#181;l&#160;of Denaturation Buffer and incubate at RT for 3 min. Combine the dilution with 45 &#181;l&#160;of Neutralization Buffer.</li>
		<li>Prepare 20 &#181;l&#160;of PCR mastermix per sample. The 1.1x PCR reaction master mixture consists of 60 units/&#181;l Taq DNA polymerase, 11 mM Tris-HCl, 55mM KCl, 1.65 mM MgCl2, 0.22 mM dNTP, 1.1x dsDNA-binding dye, 1.1x reference dye, and 550 nM each primer.</li>
		<li>Combine 2 &#181;l&#160;of diluted template and 20 &#181;l&#160;of mastermix. 
		Note: The final template concentrations in the PCR reactions shown were 50 pg, 5 pg, 500 fg, 50 fg, 5 fg, 500 ag, and 0 g Lambda DNA per microliter, and were performed in triplicate.</li>
		<li>Thermocycle PCR reaction in qPCR machine with the following program. Run 40 cycles of 95 &#176;C for 30 sec, 57 &#176;C for 20 sec, 72 &#176;C for 30 sec before 2 min at 72 &#176;C for 2 min and holding at 4 &#176;C.</li>
		<li>Normalize dsDNA binding dye fluorescence using reference dye fluorescence prior to further analysis.</li>
	</ol>
	</li>
	<li>Prepare MDA reaction.
	<ol>
		<li>Dilute samples as described in step 8.1.1.</li>
		<li>Prepare 11 &#181;l&#160;of master mixture per sample on ice. The 2x MDA reaction master mixture consists of 2x dsDNA-binding dye, 2x reference dye, 50 &#181;M randomized oligo, 2 mg/ml&#160;BSA, 2x phi29 DNA Polymerase Reaction Buffer, 4.8 mM dNTPs, nuclease-free water, and 40 &#181;g/ml&#160;Phi29 DNA Polymerase.</li>
		<li>Add the Phi29 DNA polymerase to 40 &#181;g/ml&#160;last to ensure the polymerase does not encounter pH levels much higher or lower than 7.5. Mix well.</li>
		<li>Denature 3.3 &#181;L of diluted template with 3.3 &#181;l&#160;of Denaturation Buffer in a qPCR tube. Incubate at RT for 3 min. Add 3.3 &#181;l&#160;of Neutralization Buffer.</li>
		<li>Combine 10 &#181;L of denatured template and 10 &#181;l&#160;of master mixture. Mix well.</li>
		<li>Run MDA reaction in qPCR machine with the following program.</li>
		<li>Hold at 30 &#176;C for 4 hr, measure fluorescence every 7.5 min.</li>
		<li>Inactivate at 75 &#176;C for 1 min.</li>
		<li>Normalize dsDNA binding dye fluorescence using reference dye fluorescence. Further normalize by the maximum fluorescence for each sample.</li>
	</ol>
	</li>
</ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picoinjection+enables+digital+detection+of+RNA+with+droplet+rt-PCR.">Picoinjection enables digital detection of RNA with droplet rt-PCR.</a> PLoS One. 8 (4), e62961 (2013).</li><li>Dube, S., Qin, J., Ramakrishnan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mathematical+analysis+of+copy+number+variation+in+a+DNA+sample+using+digital+PCR+on+a+nanofluidic+device.">Mathematical analysis of copy number variation in a DNA sample using digital PCR on a nanofluidic device.</a> PLoS One. 3 (8), e2876 (2008).</li><li>Shen, F., 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=Multiplexed+quantification+of+nucleic+acids+with+large+dynamic+range+using+multivolume+digital+RT-PCR+on+a+rotational+SlipChip+tested+with+HIV+and+hepatitis+C+viral+load.">Multiplexed quantification of nucleic acids with large dynamic range using multivolume digital RT-PCR on a rotational SlipChip tested with HIV and hepatitis C viral load.</a> J Am. Chem. Soc. 133 (44), 17705-17712 (2011).</li><li>Link, D. R., Anna, S. L., Weitz, D. A., Stone, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometrically+mediated+breakup+of+drops+in+microfluidic+devices.">Geometrically mediated breakup of drops in microfluidic devices.</a> Phys. Rev. Lett. 92 (5), 054503 (2004).</li><li>Nisisako, T., Torii, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+large-scale+integration+on+a+chip+for+mass+production+of+monodisperse+droplets+and+particles.">Microfluidic large-scale integration on a chip for mass production of monodisperse droplets and particles.</a> Lab Chip. 8 (2), 287-293 (2008).</li><li>Romanowsky, M. B., Abate, A. R., Rotem, A., Holtze, C., Weitz, D. A. <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+production+of+single+core+double+emulsions+in+a+parallelized+microfluidic+device.">High throughput production of single core double emulsions in a parallelized microfluidic device.</a> Lab Chip. 12 (4), 802-807 (2012).</li></ol>

The Broad Institute may move to file a patent application that includes aspects of this work.

Digital assays are powerful methods that enable detection of rare cells and counting of individual of nucleic acid molecules. However, digital assays are still not routinely applied in analytical laboratories, due in part to the cost of specialized equipment associated with commercially available methods. Here we describe an endpoint digital assay for quantifying nucleic acids with a simplified analog readout using a standard real-time quantitative PCR machine. This method is quick to set-up, and readout is much simpler ...

Digital assays for nucleic acid quantification (digital PCR)1-4 and base order (sequencing) are strongly impacting the life sciences and medicine. Digital assays provide quantification of molecular counts on an absolute scale (not relative to a control), providing high sensitivity, enabling facile comparisons across experiments, and crucially, enabling the construction of large databases containing comparable data5 (Table 1).
Over the last 15 years, whole genome amplification (WGA) emerged alongside PCR as a general tool for nucleic acid amplification. Like PCR, WGA is useful for analytical and preparative applications by amplifying minute sample elements up to a level that can be easily detected or used for subsequent analyses like base sequencing. Unlike PCR, WGA is not specific to a particular DNA locus, rather allowing amplification of all sequences in the sample, including unknown sequences. This fundamental difference between PCR and WGA makes the methods complementary to one another and gives rise to different challenges in their application.
The high yield of WGA reactions6 enables routine amplification of genomic DNA from single molecules7, single cells8 and other low-biomass samples9 for quantification or further analysis. The major challenges associated with WGA chemistry are its extreme sensitivity to contaminants and the uneven amplification across individual template molecules6. However, WGA is gaining popularity as single-cell sequencing has emerged as the &#8220;killer application&#8221; of WGA technology10, and template quantification by WGA is important in&#160;many fields of application7.
Instrumentation for digital nucleic acid quantification has been previously described in a variety of valved and valveless microfluidic formats11-14, including droplet-based assays15,16 (Table 2). However, commercial microfluidic systems for digital analysis require specialized equipment for reaction setup and product detection17. Custom valved microfluidics are flexible, but require precision microfabrication and pneumatic control systems18. While it is relatively simple to make monodisperse micro-droplets for emulsion-based digital assays19,20, digital readout is technically burdensome, requiring either large-scale wide-field imaging (similar to popular next-generation sequencing technologies)21,22 or high-speed flow-based droplet detection23-25. Ideally, a digital assay would be simple from set-up to readout, reducing the need for complex instrumentation and allowing large numbers of samples to be read out quickly. Here we describe a simplified method for readout of digital droplet assays that uses a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
While the new approach can be applied to digital PCR assays, it is particularly advantageous for digital WGA assays for which analog real-time amplification assays (that give excellent results for PCR) are problematic. WGA is commonly applied to samples with template molecules that are heterogeneous in sequence, length, and base content. These different template molecules are amplified at different rates6, necessitating the use of a reference (&#8220;standard&#8221;) sample with matching characteristics. Often, no such standard is available, or the characteristics of the incoming samples are unknown. The heterogeneity of the input material and length-dependent property of WGA chemistries26 also complicate the interpretation of results by creating ambiguity in what is being quantified--the input mass, input number of molecules, a combination of the two, or neither. Finally, the sequence-non-specificity renders quantitative multiple displacement amplification (MDA) more sensitive to contamination than quantitative PCR since contaminant molecules of any sequence have a potential to interfere. Microfluidic digital assays address contamination by segregating template molecules and reducing reaction volumes such that fewer contaminants are sampled.
Here we use a popular isothermal WGA method, MDA27. Of note, several other WGA chemistries including PicoPlex and MALBAC28 depend crucially on initial isothermal strand displacement steps. Isothermal steps exacerbate the challenge of applying analog real-time assays for quantitative WGA. WGA can neither be entirely prevented during setup, leading to unwanted variable pre-amplification, nor discretized (&#8220;cycled&#8221;) in a way where replication of heterogeneous molecules can be driven to completion and stopped prior to the next cycle like PCR11 (Figure 1). A digital assay format for WGA accomodates typical&#160;reaction setup procedures due to the segregation of each molecule&#160;for enumeration at the assay endpoint (so pre-amplification does not affect the results) original readout&#160;means accuracy would be largely independent of variation in amplification efficiency.
The assay depends on droplets produced in oil with uniform volumes, as the signal level per droplet at the assay endpoint will depend on the droplet volume, and we do not want the consistency of results to depend on averaging across a distribution of droplet sizes. Making monodisperse droplets is now a standard procedure (about 3,500 monodisperse droplet papers have been published since 2013), but requires microfluidic instrumentation25. In fact, more than six companies have developed independent commercial products that rely on production of such droplets, and droplet-making microfluidic chips are commercially available23,29. For this study, we used custom microfluidic devices produced in-house (see Protocol). Syringe pumps to drive flow through the devices are also commercially available, but alternatively, can be substituted with a single disposable syringe for vacuum-driven flow to reduce costs30.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Evagreen Dye</td>
			<td>Biotium</td>
			<td>31000</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>New England Biotechnologies</td>
			<td>B9000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phi29 DNA Polymerase Reaction Buffer</td>
			<td>New England Biotechnologies</td>
			<td>B0269S</td>
			<td>Vortex periodically until aggregate dissolves</td>
		</tr>
		<tr>
			<td>Lambda DNA</td>
			<td>New England Biotechnologies</td>
			<td>N3011S</td>
			<td>Heated to 50C and quenched on ice prior to dilution</td>
		</tr>
		<tr>
			<td>Randomized MDA oligos</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>5&#39;-NNNNN*N-3&#39;</td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>5&#8217;-CGGCAAACGGGAATGAAACGCC-3&#8217; and 5&#8217;-TGCGGCAAAGACAGCAACGG-3&#8217;</td>
		</tr>
		<tr>
			<td>UltraPure Nuclease-free water</td>
			<td>Life Technologies</td>
			<td>10977-015</td>
			<td>UV-treated for 30m in Stratalinker 2400 before use (optional)</td>
		</tr>
		<tr>
			<td>ROX reference dye</td>
			<td>Life Technologies</td>
			<td>12223-012</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR optical strip caps</td>
			<td>Life Technologies</td>
			<td>4323032</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>Agilent Technologies</td>
			<td>401428</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP (25uM each)</td>
			<td>Agilent Technologies</td>
			<td>200415</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler - Mx3000P qPCR system</td>
			<td>Agilent Technologies</td>
			<td>401403</td>
			<td></td>
		</tr>
		<tr>
			<td>Barrier pipette tips</td>
			<td>VWR</td>
			<td>89003-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-rad oil for evagreen</td>
			<td>Bio-rad</td>
			<td>186-4005</td>
			<td></td>
		</tr>
		<tr>
			<td>JumpStart Taq ReadyMix</td>
			<td>Sigma-Aldrich</td>
			<td>P2893-100RXN</td>
			<td></td>
		</tr>
	</tbody>
</table>

simple bulk readout of digital nucleic acid quantification assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

While conventional bulk/real-time readouts can be used for both quantitative PCR and quantitative WGA assays (Figure 1), digital quantitative assays provide advantages (Table 1). In the method described, we read out digital assays in micro-droplet format with a simple bulk endpoint measurement (Figure 2). While this method is broadly applicable, we focus on quantitative WGA (MDA) because this method presents special challenges for conventional real-time assays.
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Watch this Scientific Journal Video about Simple Bulk Readout of Digital Nucleic Acid Quantification Assays at JoVE.com

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

simple-bulk-readout-of-digital-nucleic-acid-quantification-assays

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Biology

8.2K Views.  Broad Institute. We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy. 

Video: Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

FtsZ Polymerization Assays: Simple Protocols and Considerations

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long filaments in the presence of GTP in vitro, and polymerization is regulated by several accessory proteins. FtsZ polymerization has been extensively studied in vitro using basic methods including light scattering, sedimentation, GTP hydrolysis assays and electron microscopy. Buffer conditions influence both the polymerization properties of FtsZ, and the ability of FtsZ to interact with regulatory proteins. Here, we describe protocols for FtsZ polymerization studies and validate conditions and controls using Escherichia coli and Bacillus subtilis FtsZ as model proteins. A low speed sedimentation assay is introduced that allows the study of the interaction of FtsZ with proteins that bundle or tubulate FtsZ polymers. An improved GTPase assay protocol is described that allows testing of GTP hydrolysis over time using various conditions in a 96-well plate setup, with standardized incubation times that abolish variation in color development in the phosphate detection reaction. The preparation of samples for light scattering studies and electron microscopy is described. Several buffers are used to establish suitable buffer pH and salt concentration for FtsZ polymerization studies. A high concentration of KCl is the best for most of the experiments. Our methods provide a starting point for the in vitro characterization of FtsZ, not only from E. coli and B. subtilis but from any other bacterium. As such, the methods can be used for studies of the interaction of FtsZ with regulatory proteins or the testing of antibacterial drugs which may affect FtsZ polymerization.

Polymerization of FtsZ is essential for bacterial cell division. In this report, we detail simple protocols to monitor FtsZ polymerization activity and discuss the influence of buffer composition. The protocols can be used to study the interaction of FtsZ with regulatory proteins or antibacterial drugs that affect FtsZ polymerization.

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

The Lactate Dehydrogenase Sequestration Assay &amp;#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce musculature-driven forms of locomotion: peristalsis or scrunching. While peristalsis results from a ciliary defect, scrunching is independent of cilia function and is a specific response to certain stimuli, including amputation, noxious temperature, extreme pH, and ethanol. Thus, these two musculature-driven gaits are mechanistically distinct. However, they can be difficult to distinguish qualitatively. Here, we provide a protocol for inducing scrunching using various physical and chemical stimuli. We detail the quantitative characterization of scrunching, which can be used to distinguish it from peristalsis and gliding, using freely available software. Since scrunching is a universal planarian gait, albeit with characteristic species-specific differences, this protocol can be broadly applied to all species of planarians, when using appropriate considerations. To demonstrate this, we compare the response of the two most popular planarian species used in behavioral research, Dugesia japonica and Schmidtea mediterranea, to the same set of physical and chemical stimuli. Furthermore, the specificity of scrunching allows this protocol to be used in conjunction with RNA interference and/or pharmacological exposure to dissect the molecular targets and neuronal circuits involved, potentially providing mechanistic insight into important aspects of nociception and neuromuscular communication.

Freshwater planarians exhibit three gaits (gliding, peristalsis, and scrunching) that are distinguishable by quantitative behavioral analysis. We describe a method to induce scrunching using various noxious stimuli, quantification thereof, and distinction from peristalsis and gliding. Using gene knockdown, we demonstrate the specificity of scrunching as a quantitative phenotypic readout.

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce ...