Name: bidirectional retroviral integration site pcr methodology and quantitative data analysis workflow
Uploaded: 2017-06-14T15:00:00
Description: Watch this Scientific Journal Video about Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow at JoVE.com

Funding was provided by the National Institutes of Health Grants R00-HL116234, U19 AI117941, and R56 HL126544; the National Science Foundation Grant DMS-1516675; the National Research Foundation of Korea (NRF-2011-0030049, NRF-2014M3C9A3064552); and the KRIBB initiative program.

1. Generating Upstream (left)- and Downstream (right)-junction Sequence Libraries
<ol>
	<li>DNA linker preparation:
	<ol>
		<li>Prepare a 10 &#181;L linker DNA solution by adding 2 &#181;L of 100 &#181;M LINKER_A oligos (final: 20 &#181;M), 2 &#181;L of 100 &#181;M LINKER_B oligos (final: 20 &#181;M), 2 &#181;L of 5 M NaCl (final: 1M), and 4 &#181;L of nuclease-free water in a PCR tube. See Table 1 for the linker sequences.</li>
		<li>Incubate the linker DNA solution at 95 &#176;C for ...

<ol>
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The bidirectional assay enables the simultaneous analysis of both the upstream (left) and downstream (right) vector-host DNA junction sequences and is useful in a number of gene therapy, stem cell, and cancer research applications. The use of GTAC-motif enzymes (RsaI and CviQI) and the bidirectional PCR approach significantly improves the chances of detecting an integrome (or a clonal population) when compared to previous TCGA-motif enzyme (Taq&#945;I)-based assays2,<sup class=...

Retroviruses insert their genomic DNA into the host genome at various sites. This unique property, which may contribute to the development of cancers and other forms of viral pathogenesis, has the ironic benefit of making these viruses highly amenable to cellular engineering for gene therapy and basic biology research. The viral Integration Site (IS) &#8211; the location on the host genome where a foreign DNA (virus) is integrated &#8211; has important implications for the fate of both the integrated viruses and the host cells. IS assays have been used in various biological and clinical research settings to study retroviral integration site selection and pathogenesis, cancer development, stem cell biology, and developmental biology1,2,3,4. Low detection sensitivity, poor data reproducibility, and frequent cross-contamination are among the key factors limiting the applications of IS assays to current and planned studies.
Many IS analysis technologies have been developed. Restriction enzyme-based integration site assays, including Linker-Mediated (LM) Polymerase Chain Reaction (PCR)5, inverse PCR6, and Linear-Amplification-Mediated (LAM) PCR7, are the most widely used. The use of site-specific restriction enzymes, however, generates a bias during the retrieval of the IS, allowing only a subset of integromes (a foreign DNA integrated into the host genome) in the vicinity of the restriction site to be recovered4. Assay technologies that more comprehensively assess vector IS have also been introduced in recent years. These assays employ various strategies, including Mu transposon-mediated PCR8, nonrestrictive (nr)-LAM PCR9, type-II restriction enzyme-mediated digestion10, mechanical shearing11, and random hexamer-based PCR (Re-free PCR)12, to fragment genomic DNAs and amplify IS. Current technologies have varying levels of detection sensitivity, genome coverage, target specificity, high-throughput capacity, complexity of assay procedures, and biases in detecting the relative frequencies of target sites. Given the varying qualities of the existing assays and the variety of purposes for which they can be used, the optimal assay approach should be carefully selected.
This work provides detailed experimental procedures and a computational data analysis workflow for a bidirectional assay that significantly improves detection rates and sequence quantification accuracy by simultaneously analyzing the IS upstream and downstream of the integrated target DNA (see Figure 1 for a schematic view of the assay procedures). This approach also provides the means to characterize the retroviral integration process (for example, the fidelity of target site duplication and variations in the genomic sequences of upstream and downstream insertions). Other bidirectional methods have been used primarily for cloning and sequencing both ends of the target DNA11,13,14. This assay is extensively optimized for the high-throughput and reproducible quantification of vector-marked clones, using the well-established LM-PCR method and computational analysis mapping, and for quantifying both upstream and downstream junctions. Bidirectional analysis with the Taq&#945;I enzyme has proven useful for high-throughput clonal quantification in stem cell gene therapy preclinical studies2,15. This paper describes a modified method using a more frequent cutter (RsaI/CviQI - motif: GTAC) that doubles the chances of detecting integromes compared to a Taq&#945;I-based assay. Detailed experimental and data analysis procedures that use GTAC motif enzymes for lentiviral (NL4.3 and its derivatives) and gamma-retroviral (pMX vectors) vector IS analysis are described. The oligonucleotides used in the assay are listed in Table 1. An in-house programming script for IS sequence analysis is provided in the supplemental document.

<table border="0" cellpadding="0" cellspacing="0" width="1004">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Thermostable DNA polymerase</td>
			<td style="width:208px;">Agilent</td>
			<td style="width:119px;">600424</td>
			<td style="width:347px;">PicoMaxx Polymerase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermostable DNA polymerase buffer</td>
			<td style="width:208px;">Agilent</td>
			<td>600424</td>
			<td style="width:347px;">PicoMaxx Polymerase buffer</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Deoxynucleotide (dNTP) solution mix</td>
			<td style="width:208px;">New England Biolabs</td>
			<td>N0447L</td>
			<td>dNTP solution mix (10mM each)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR tubes</td>
			<td style="width:208px;">VWR International</td>
			<td style="width:119px;">53509-304</td>
			<td style="width:347px;">PCR Strip Tubes With Individual Attached Caps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">2ml microcentrifuge tube</td>
			<td style="width:208px;">Molecular Bioproducts</td>
			<td style="width:119px;">3453</td>
			<td>microcentrifuge tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">PCR purification kit</td>
			<td style="width:208px;">Qiagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">RsaI&#160;</td>
			<td style="width:208px;">New England Biolabs</td>
			<td>R0167L</td>
			<td>restriction enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">CviQI</td>
			<td style="width:208px;">New England Biolabs</td>
			<td>R0639L</td>
			<td>restriction enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Buffer A</td>
			<td style="width:208px;">New England Biolabs</td>
			<td>B7204S</td>
			<td>NEB CutSmart buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA Polymerase I large (klenow) fragment&#160;</td>
			<td style="width:208px;">New England Biolabs</td>
			<td>M0210L</td>
			<td>Blunting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">streptavidin beads solution</td>
			<td>Invitrogen&#160;</td>
			<td>60101</td>
			<td>Dynabeads kilobaseBINDER kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Binding Solution</td>
			<td>Invitrogen&#160;</td>
			<td>60101</td>
			<td>Dynabeads kilobaseBINDER kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Washing Solution</td>
			<td>Invitrogen&#160;</td>
			<td>60101</td>
			<td>Dynabeads kilobaseBINDER kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">magnetic stand</td>
			<td style="width:208px;">ThermoFisher</td>
			<td>12321D</td>
			<td>DynaMag&#8482;-2 Magnet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">T4 DNA ligase</td>
			<td style="width:208px;">New England Biolabs</td>
			<td>M0202L</td>
			<td>T4 DNA ligase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">10X T4 DNA ligase buffer</td>
			<td style="width:208px;">New England Biolabs</td>
			<td>B0202S</td>
			<td>T4 DNA ligase reaction buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">5X T4 DNA ligase buffer</td>
			<td style="width:208px;">Invitrogen&#160;</td>
			<td>46300-018</td>
			<td>T4 DNA ligase buffer with polyethylene glycol-8000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV-Vis spectrophotometer</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td>S06497</td>
			<td style="width:347px;">Nanodrop 2000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">pvuII</td>
			<td style="width:208px;">new England Biolabs</td>
			<td>R0151L</td>
			<td>restriction enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">sfoI</td>
			<td style="width:208px;">new England Biolabs</td>
			<td>R0606L</td>
			<td>restriction enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buffer B</td>
			<td style="width:208px;">new England Biolabs</td>
			<td>B7203S</td>
			<td>NEB buffer 3.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Nuclease free water</td>
			<td style="width:208px;">Integrated DNA Technologies</td>
			<td>11-05-01-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Capillary electrophoresis</td>
			<td style="width:208px;">Qiagen</td>
			<td>9001941</td>
			<td style="width:347px;">QIAxcel capillary electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Veriti 96-well Fast Thermal Cycler</td>
			<td style="width:208px;">Thermo Fisher Scientific</td>
			<td>4375305</td>
			<td>PCR Instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Rotating wheel (or Roller)</td>
			<td style="width:208px;">Eppendorf</td>
			<td>M10534004</td>
			<td>Cell Culture Roller Drums</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">DNA size marker</td>
			<td style="width:208px;">Qiagen</td>
			<td>929559</td>
			<td>QX size marker (100-2,500 bp)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">DNA size marker</td>
			<td style="width:208px;">Qiagen</td>
			<td>929554</td>
			<td>QX size marker (50-1,500 bp)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">DNA alignment markers</td>
			<td style="width:208px;">Qiagen</td>
			<td>929524</td>
			<td>QX DNA Alignment Marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">genomc DNA</td>
			<td style="width:208px;">Not Available</td>
			<td style="width:119px;">Not Available</td>
			<td>Sample genomc DNA from in vivo or in vitro experiments</td>
		</tr>
	</tbody>
</table>

bidirectional retroviral integration site pcr methodology and quantitative data analysis workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

The bidirectional IS assay generated different sizes of PCR amplicons for both the upstream (left) and downstream (right) vector host junctions (Figure 2). The size of a PCR amplicon is dependent on the location of the nearest GTAC motif upstream and downstream from an integrome. The assay also produced internal DNA PCR amplicons: retroviral sequences near the polypurine tract and the primer binding site were concomitantly amplified during left- and right-jun...

Watch this Scientific Journal Video about Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow at JoVE.com

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

The overall goal of this procedure is to identify retroviral vector integration sites in the host genome and quantify the relative frequencies of clonal cell populations each sharing the same integration site. This method can help answer key questions in retroviral gene therapy, such as which part of the host genome vector has integrated, how a genetically engineered cell will repopulate after the transplant, and how they are functionally different. The main advantage of this technique is that retro-integration site sequences can be analyzed with higher accuracy and sensitivity by sequencing both upstream and downstream vector host DNA junctions simultaneously.Though this method can provide insight into the safety of vectors and gene engineered cell in gene therapy studies, it can also be applied to other basic biology studies to investigate individual cell behavior in vivo. The first step in this procedure is to determine the DNA concentration and the ratio of absorbance at 260 and 280 nanometers of the genomic DNA to be analyzed. After this, dilute one to two micrograms of the sample genomic DNA to a final volume of 170 microliters of genomic DNA solution using nuclease free water.Prepare a 200 microliter PCR reaction by mixing the following in a microcentrifuge tube. 2.5 microliters each of HIV-1 specific biotin primers, or a total of 10 microliters for four lentivirus-specific primers, 20 microliters of 10X thermostable DNA polymerase buffer, four microliters of 10 millimolar dNTPs, three microliters of thermostable DNA polymerase, and 163 microliters of genomic DNA. Divide the solution equally into four PCR tubes and carry out a single extension cycle under the following condition.94 degrees Celsius for 5 minutes, 56 degrees Celsius for 3 minutes, 72 degrees Celsius for 5 minutes, and 4 degrees C for storage. Pool all four PCR reactions into a two milliliter microcentrifuge tube. Follow the manufacturer's procedure of a PCR purification kit, and elute with 50 microliters of elution buffer.Prepare for a 100 microliter digestion by adding the following to a microcentrifuge tube. 50 microliters of DNA, 10 microliters of 10X buffer A, two microliters of RSAI enzyme, and 38 microliters of nuclease-free water. Incubate at 37 degrees Celsius in a thermal cycler for one hour.Next, add one microliter of CviQI enzyme to the reaction and incubate at 25 degrees Celsius for 30 minutes. When the RSAI and CviQI digestion is complete, proceed immediately with blunt ending. Prepare a 4.5 microliter mixture containing 2.5 microliters of DNA polymerase I, Large Fragment, and 2 microliters of 10 millimolar dNTPs.Transfer 1 microliter of the mixture to the DNA sample. The total volume will be 102 microliters. Mix well by vortexing and incubate at 25 degrees Celsius for one hour.Immediately proceed with streptavidin bead binding when the incubation is complete. Prepare the streptavidin beads by briefly vortexing the bead solution and transferring 50 microliters to a new two milliliter microcentrifuge tube. Remove the supernatant using a magnetic stand and wash the beans with 200 microliters of binding solution.Resuspend the beads in 100 microliters of binding solution and place the tube away from the magnetic stand. Transfer 100 microliters of the sample DNA to the 100 microliters of resuspended bead solution and mix carefully by pipetting to avoid any foaming of the solution. Incubate the tube at room temperature for 3 hours on a rotating wheel.Use the magnetic stand to capture the beads and discard the supernatant. Wash the beads twice in 400 microliters of washing solution and twice in 400 microliters of 1X T4 DNA ligase buffer. Resuspend the beads in 200 microliters of 1X T4 DNA ligase buffer and place it away from the magnetic stand.Linker ligation should be performed immediately after the streptavidin bead binding. Prepare a 400 microliter ligation reaction solution by mixing the following in a two milliliter microcentrifuge tube. 0.5 microliters of the DNA linker, 10 microliters of 10X T4 DNA ligase buffer, 20 microliters of 5X T4 DNA ligase buffer, 5 microliters of T4 DNA ligase, 164.5 microliters of nuclease free water, and 200 microliters of the resuspended beads.Place the reaction tube on a rotating wheel at room temperature for 3 hours. Wash the beads twice with the washing solution, and twice with 1X thermostable DNA polymerase buffer. Resuspend the beads in 50 microliters of 1X thermostable DNA polymerase PCR buffer, and place it away from the magnetic stand.To begin this procedure prepare a 200 microliter PCR reaction by adding the following to the resuspended beads. 10 microliters of one 1L-primer, 10 microliters of 1R-primer, 20 microliters of primer Link1, 10 microliters of 10X thermostable DNA polymerase buffer, four microliters of 10 milimolar dNTPs, eight microliters of thermostable DNA polymerase, and 88 microliters of nuclease-free water. Aliquot the reaction mixture into four PCR tubes and carry out PCR with the following condition.94 degrees Celsius for 2 minutes, 25 cycles of 94 degrees Celsius for 20 seconds, 56 degrees Celsius for 25 seconds, and 72 degrees Celsius for two minutes, and a final extension at 72 degrees Celsius for 5 minutes. Pool all four PCR reactions into a 2 ml microcentrifuge tube. Follow the procedure of the PCR purification kit and elute with 50 microliters of elution buffer.Determine the DNA concentration and the ratio of absorbance at 260 and 280 nanometers using a UV visible spectrophotometer. The DNA can now be stored at minus 20 degrees Celsius until ready for the next step. Since the procedures for the left junction and right junction specific amplifications are identical except for the primers used in the PCR, only the left junction amplification will be demonstrated.Prepare a 50 microliter PCR reaction by mixing the following in a PCR tube. Five microliters of DNA, five microliters of 10 micromolar 2L-primer, five microliters of 10 micromolar primer Link2, 5 microliters of 10X thermostable DNA polymerase buffer, one microliter of 10 milimolar dNTPs, two microliters of thermostable DNA polymerase, and 27 microliters of nuclease-free water. Carry out PCR with the following condition.94 degrees Celsius for 3 minutes, 8 to 15 cycles of 94 degrees Celsius for 20 seconds, 56 degrees Celsius for 25 seconds, and 72 degrees Celsius for two minutes, and a final extension at 72 degrees Celsius for 5 minutes. Use the PCR purification kit to purify the DNA and elute with 50 microliters of elution buffer. Determine the DNA concentration and ratio of absorbance at 260 and 280 nanometers.The last step in generating left and right junction sequence libraries is to analyze the PCR Amplicon length variations by performing capillary electrophoresis. This representative result shows varying lengths of PCR Amplicons in lentiviral vector integrations sites for both the upstream and downstream vector host junctions. The DNA size marker is in the M lane.After sequencing the PCR Amplicons, computational integration site sequence analysis is performed using an in-house programming script. The bi-directional integration site assay generates different sizes of PCR Amplicons for both the upstream and downstream vector host junctions visualized by capillary electrophoresis. A comparison of percent integration in genes Alu and L1 repeats of lentiviral vectors in humanized mouse repopulating cells within silico generated random integration events revealed that integration sites are significantly enriched in genes.The relative detection frequencies of vector integromes were calculated using only the sequence counts of integration site junctions generating smaller than 500bp PCR Amplicons. Approximately 77%of the vectors could be quantitatively analyzed by the bi-directional approach. In the strategy for clonal quantification we presume that each individual clone shares the same vector integrome.The relative frequencies of the left and right junctions are combined to represent the relative quantities of the clonal populations, or QVI. This color scheme shows the relative frequencies of 44 QVI clones in humanized mouse repopulating cells. Based on in silico 10, 000 random integration analysis, a 1.25 fold overestimation of clonal frequencies is expected when using GTAC motif enzymes, and a 2.56 overestimation is expected when using a TCGA motif enzyme.Once mastered, this technique can be divided in parts and completed in two days, if performed properly. While attempting this procedure it is important to remember that PCR steps are extremely sensitive, and precautions should be take to avoid any contamination. This technique has paved the way to examining the safety of therapeutic retroviral vectors, and to explore long-term repopulation patterns of hemotopoietic stem cell clones following transplant in rhesus macaques.

bidirectional-retroviral-integration-site-pcr-methodology

Research

JoVE Journal

Genetics

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (&#916;S) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (&#945;) or a truncated transactivation domain with 7 unique residues (&#946;). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (S&#945;, S&#946;, &#916;S&#945; and &#916;S&#946;) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites.

Tandem splicing events occur at sites less than 12 nucleotides apart. Quantifying ratios of such splice variants is feasible using an absolute quantitative PCR approach. This manuscript describes how splice variants of the gene STAT3, in which two splicing events results in Serine-701 inclusion/exclusion and &#945;/&#946; C-termini, can be quantified.

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 ...

Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been investigated in various organs and tissues in rat. However, standard methods for the purification of miRNAs and detection of their expression in rat peritoneal membrane have not been well established. We have developed an effective and reliable method to purify and quantify miRNAs using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in rat peritoneal membrane. This protocol consists of four steps: 1) purification of peritoneal membrane sample; 2) purification of total RNA including miRNA from peritoneal membrane sample; 3) reverse transcription of miRNA to produce cDNA; and 4) qRT-PCR to detect miRNA expression. Using this protocol, we successfully determined that the expression of six miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p) increased significantly in the peritoneal membrane of a rat peritoneal fibrosis model compared with those in control groups. This protocol can be used to study the profile of miRNA expression in the peritoneal membrane of rats in many pathological conditions.

Here we present a protocol for the detection of microRNA expression in rat peritoneal membrane using quantitative real-time reverse-transcription polymerase chain reaction. This method is suitable for studying the microRNA expression profile in rat peritoneal membrane in several pathological conditions.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.
The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells.

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing high-throughput short-read DNA sequencing. The resulting sequence datasets can not only track what genes in a population that are enriched during selection for positive yeast 2-hybrid interactions, but also give detailed information about the relevant subdomains of proteins sufficient for interaction. Here, we describe a full suite of stand-alone software programs that allow non-experts to perform all the bioinformatics and statistical steps to process and analyze DNA sequence fastq files from a batch yeast 2-hybrid assay. The processing steps covered by these software include: 1) mapping and counting sequence reads corresponding to each candidate protein encoded within a yeast 2-hybrid prey library; 2) a statistical analysis program that evaluates the enrichment profiles; and 3) tools to examine the translational frame and position within the coding region of each enriched plasmid that encodes the interacting proteins of interest.

Deep sequencing of yeast populations selected for positive yeast 2-hybrid interactions potentially yields a wealth of information about interacting partner proteins. Here, we describe the operation of specific bioinformatics tools and customized updated software to analyze sequence data from such screens.

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing ...

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis

Yersinia ruckeri is an important pathogen of farmed salmonids worldwide, but simple tools suitable for epizootiological investigations (infection tracing, etc.) of this bacterium have been lacking. A Multi-Locus Variable-number tandem-repeat Analysis (MLVA) assay was therefore developed as an easily accessible and unambiguous tool for high-resolution genotyping of recovered isolates. For the MLVA assay presented here, DNA is extracted from cultured Y. ruckeri samples by boiling bacterial cells in water, followed by use of supernatant as template for PCR. Primer-pairs targeting ten Variable-number tandem-repeat (VNTR) loci, interspersed throughout the Y. ruckeri genome, are distributed equally amongst two five-plex PCR reactions running under identical cycling conditions. Forward primers are labelled with either of three fluorescent dyes. Following amplicon confirmation by gel electrophoresis, PCR products are diluted and subjected to capillary electrophoresis. From the resulting electropherogram profiles, peaks representing each of the VNTR loci are size-called and employed for calculating VNTR repeat counts in silico. Resulting ten-digit MLVA profiles are then&#160;used to generate Minimum spanning trees enabling epizootiological evaluation by cluster analysis. The highly portable output data, in the form of numerical MLVA profiles, can rapidly be compared across labs and placed in a spatiotemporal context. The entire procedure from cultured colony to epizootiological evaluation may be completed for up to 48 Y. ruckeri isolates within a single working day.

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis

The Multi-Locus Variable-number tandem-repeat Analysis (MLVA) assay presented here enables inexpensive, robust and portable high-resolution genotyping of the fish-pathogenic bacterium Yersinia ruckeri. Starting from pure cultures, the assay employs multiplex PCR and capillary electrophoresis to produce ten-loci MLVA profiles for downstream applications.

Yersinia ruckeri is an important pathogen of farmed salmonids worldwide, but simple tools suitable for epizootiological investigations (infection tracing, ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named &#8216;Worm-to-CT&#8217;, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly ...