This protocol development was supported by the Bill and Melinda Gates Foundation Grand Challenges in Global Health grant 37774. Real-time PCR reagents and advice were provided by Abbott Molecular Inc. Des Plaines, IL. 8E5-LAV cells were provided by the Virology Quality Assurance Laboratory, Rush Presbyterian; St. Luke's Medical Center. HIV-1 negative blood was provided by Core Lab, NorthShore University HealthSystems, Evanston, IL. Thanks to Mark Fisher for photography help.

Ethics statement: The whole blood specimens used in this study are not considered to be research involving human subjects. The specimens were obtained for clinical diagnostic purposes, which were satisfied, and the remaining portion of these specimens was provided for the FINA research assay. The specimens were coded such that the investigators were not able to readily ascertain the identity of individuals.
1. Preparation of FINA Sample Preparation Module
<ol>
	<li>Prepare blotting pad by cutting a 35 x 35 mm2 of a 2.6 mm thick 100% cotton pad. Cut one pad per specimen.</li>
	<li>Prepare plastic paraffin film with a paper backing tape by a cutting piece of paraffin film (25 mm (h) by 50 mm (w)) and then punching a 7.14 mm diameter hole in the center of the tape using a hammer driven hole punch. Prepare one tape per specimen.</li>
	<li>Prepare a bound glass blood separator membrane (capture membrane) disk by punching a 8.35 mm diameter circle using a hammer driven hole punch. Punch one disk per specimen. The disk has a particle retention capability of 2.3 &#181;m.</li>
	<li>Assemble the FINA sample preparation module by placing the capture disk in the center of the blotting pad using forceps. Place the paraffin film tape over the capture disk so that the hole of the paraffin tape leaves the center of the capture disk exposed.</li>
	<li>Ensure maximum contact between the disk and the blotting pad by stretching the paraffin tape slightly to cover the blotting pad and pressing the paraffin tape firmly to stick it to the blotting pad around all edges of the disk.</li>
	<li>Make as many modules as needed for the experiment or stockpile for future use.</li>
</ol>
2. Preparation of qPCR Tube
<ol>
	<li>Prepare a 5.1 mm tape disk that will anchor the cell capture membrane to the side of the qPCR tube to prevent the membrane from blocking the fluorescence detection by punching a circle of double-coated polyester diagnostic tape with a hammer driven punch from a sheet of the tape. Prepare one tape disk per specimen.</li>
	<li>Remove one side of the tape&#39;s sticker liner. Place the tape into a 200 &#181;l PCR tube, sticking it onto one side and toward the bottom of the tube. Remove the second sticker liner. The tube is now ready to receive prepared disk.</li>
	<li>Produce as many tubes as needed for the experiment or stockpile for future use.</li>
</ol>
3. Contrive Blood Specimen
Note: If a genuine test specimen is being prepared, proceed to step 4.
<ol>
	<li>Thaw 8e5-LAV cells 15 harboring a single copy of the HIV-1 provirus obtained from the Virology Quality Assurance Laboratory (VQA; Rush Presbyterian/St. Luke&#39;s Medical Center, Chicago, IL). 
	Note: They are stored as frozen cell pellets at a concentration of 4,000 cells/&#181;l. Cell count was verified as described previously 9.</li>
	<li>Prepare serial dilution in freezing medium (90% fetal bovine serum, 10% dimethyl sulfoxide) to concentrations ranging from 1 to 400 cells/&#181;l.</li>
	<li>Pipet 10 &#181;l cells from each of the serial dilutions into a microcentrifuge tube. Add 100 &#181;l of fresh HIV-1 negative EDTA treated whole blood sample to each tube to create a standard curve of 8e5-LAV copy numbers 10-4,000. Mix by gently flicking the bottom of the tube 5 times.</li>
</ol>
4. Perform FINA Extraction
<ol>
	<li>Lyse blood cells by adding Triton-X100) to a final concentration of 1% (11 &#181;l 10% Triton-X100 to 110 &#181;l whole blood and cells from step 3.3).</li>
	<li>Mix by gently flicking the bottom of the tube 5 times. The blood should turn translucent red.</li>
	<li>Pipet all of the blood specimen onto the capture disk. 
	Note: A water tight seal between the paraffin tape and the capture membrane ensures that the blood flows through the membrane, and good contact between the capture membrane and blotting pad assembly ensures rapid sample wicking.</li>
	<li>Allow sample to soak through the filter. 
	Note: The blood lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the capture disk. The lysate has soaked into the disk when it appears matte.</li>
	<li>Add 600-1,000 &#181;l 10 mM NaOH drop-wise onto the capture disk. 
	Note: The wash buffer can also be added as a bulk addition of 600 &#181;l. The buffer will form a droplet on the hydrophobic paraffin tape and wick through the hole to the disk in approximately 20 sec. The filter will change from red to white indicating clearance of hemoglobin.</li>
	<li>Separate the disk from the blotting pad with forceps and apply the disk to the tape in the prepared PCR tube. If there is any red color left on the filter add 50-100 &#181;l of wash buffer to clear the filter before placing in the tube. 
	Note: Infrequently, excess pressure applied during preparation of the FINA modules results in partitioning of the membrane layers during separation from the blotting pad. Discard these disks and prepare a fresh sample.</li>
	<li>After the filter is placed in the PCR tube, analyze the samples right away. Alternatively, dry overnight in a box containing calcium sulfate desiccant, then cap and place in a foil pouch with silica desiccant and store until ready for analysis.</li>
</ol>
5. qPCR Reaction
<ol>
	<li>Prepare 100 &#181;l qPCR master mix per reaction using HIV specific primers and probes as described previously 9,11. Include an internal amplification control to monitor for the presence of amplification inhibitors and to verify that a negative sample is a true negative. Be sure that filter is completely covered with master mix and that the filter stays fixed to side of tube throughout the reaction.</li>
	<li>Perform amplification using a real-time PCR device as previously described 9.</li>
</ol>

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+low+cost+point-of-care+viscous+sample+preparation+device+for+molecular+diagnosis+in+the+developing+world;+an+example+of+microfluidic+origami.">A low cost point-of-care viscous sample preparation device for molecular diagnosis in the developing world; an example of microfluidic origami.</a> Lab Chip. 12 (1), 174-181 (2012).</li><li>Linnes, J. C., 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=Paper-based+molecular+diagnostic+for.">Paper-based molecular diagnostic for.</a> RSC Adv. 4 (80), 42245-42251 (2014).</li><li>Rodriguez, N. 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The authors have nothing to disclose.

EID linked to rapid treatment access has been demonstrated to reduce infant mortality due to HIV infection 16. Because of the persistence of maternal antibodies in an infant&#39;s blood, rapid HIV antibody tests have limited utility in determining the status of HIV-exposed infants. The WHO recommends that all infants born to HIV-1 positive mothers should be tested at 4-6 weeks of age, using a virological test 17. We have reported the development of an assay for the detection and quantitation of HIV-...

Several reports have discussed the development of paper- or membrane-based extraction methods for use in point-of-care (POC) devices 1-5 with the aim of making the exquisite sensitivity and specificity of molecular diagnostics available to all. The World Health Organization (WHO) Sexually Transmitted Diseases Diagnostics Initiative coined the term ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free and Delivered to those who need it) to describe the ideal characteristics of a POC test 6. Of these guidelines, the equipment-free characteristic is particularly challenging to achieve for molecular diagnostics. However, every innovation in the field will advance the goal of reaching those in most need, and there is hope for near-term improvements in test performance by adapting existing technology 7.
Here we describe a simple protocol for extracting DNA from whole blood that does not require complex chemistry or laboratory instrumentation. The FINA (filtration isolation of nucleic acids) sample preparation method was originally developed to extract leukocyte DNA from whole blood in order to detect the HIV-1 provirus as part of a sample-to-answer point-of-care (POC) quantitative PCR (qPCR) early infant diagnostic (EID) platform for use in limited resource settings 8-11. FINA extraction differs from conventional purification methods which use silica membranes or silica-coated paramagnetic particles to reversibly bind DNA in the presence of chaotropic agents 12. Instead, FINA uses vertical filtration via a separation membrane to extract cellular DNA from whole blood directly. The membrane containing the entrapped DNA can be placed directly in a PCR tube and either immediately used in a PCR reaction or air dried for later amplification 9. No chaotropic agents, phenol, or alcohols are used in the sample extraction, eliminating the extensive washing steps needed to remove potent qPCR inhibitors derived from the extraction process 13,14.
The FINA membrane can capture either cells 9 or genomic DNA liberated by cell lysis 11 before the specimen is added to the membrane. For the cell capture, whole blood is added directly to the membrane. The cells are subsequently lysed in the membrane by adding 10 mM NaOH. The advantage to this method is that it involves only 3 steps: 1) sample addition; 2) cell lysis/wash and 3) filter disk placement in qPCR tube. The drawback to this method is that the membrane can only hold a defined number of cells directly proportional to the diameter of the membrane disk. To reach the limit of detection required for EID, 100 &#181;l of whole blood is required for sample input which entails a filter that is too large to be placed in a qPCR tube. Lysing the blood cells with detergent before adding the sample to the collection membrane adds a processing step, but allows the use of a smaller filter for the same sample size. We were able to demonstrate high reproducibility, single copy detection, and quantification of as little as 10 copies of HIV-1 proviral DNA from 100 &#181;l of blood using this test configuration 11.
In this report, we describe the FINA protocol as originally developed for laboratory use. The membrane/filter sandwich known as the FINA sample preparation module can be prepared in large batches and stockpiled for later use. When specimens are to be extracted this process takes 2 min and can be performed in varying size batches. The qPCR can be run immediately or the filters containing the embedded DNA can be stored until it is convenient to perform qPCR. This method is very convenient for routine analysis of specimens in both low and high resource settings.

<table border="0" cellpadding="0" cellspacing="0" width="860">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Fusion 5</td>
			<td style="width:179px;">GE Healthcare Life Sciences</td>
			<td style="width:104px;">8151-9915</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">707 blotting pad</td>
			<td>VWR International</td>
			<td>28298-014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PARAFILM M</td>
			<td>VWR International</td>
			<td>52858-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3M Double-Coated Polyester Diagnostic Tape&#160;</td>
			<td>3M Medical Specialties</td>
			<td>9965</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 &#181;l qPCR strip tubes</td>
			<td>Agilent</td>
			<td>401428</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">optical strip caps</td>
			<td>Agilent</td>
			<td>401425</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mx3005p qPCR System</td>
			<td>Agilent</td>
			<td>401456</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td>A.C.S. Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td>BioXtra</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td>for molecular biology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fetal bovine serum, certified, U.S. origin</td>
			<td>Thermo Fisher Scientific</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hammer driven small hole punch 3/16&#34; hole diameter (5.1 mm)</td>
			<td>McMaster Carr</td>
			<td>3424A16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hammer driven small hole punch 1/4&#34; hole diameter (7.14 mm)</td>
			<td>McMaster Carr</td>
			<td>3424A19</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hammer driven small hole punch 5/16&#34; hole diameter (8.35 mm)</td>
			<td>McMaster Carr</td>
			<td>3424A23</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The workflow for extracting proviral DNA from whole blood spiked with 8e5-LAV cells is shown in Figure 1. Figure 2 shows the FINA sample module and prepared qPCR tube. This method allows for efficient amplification of HIV-1 provirus from 8e5-LAV cells at different copy numbers, as shown in the standard curve of contrived specimens (Figure 3). PCR had an efficiency of 103%, as calculated from Efficiency = -1+10(-1/slope). Equati...

Watch this Scientific Journal Video about Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method at JoVE.com

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

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 ...

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18.0K Views.  Northwestern University. 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.

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

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 ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

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 ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

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.

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 ...

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 ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

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

Summary

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Chapters in this video

NanoDrop Microvolume Quantitation of Nucleic Acids

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

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

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix