This work was funded by the Wellcome Trust through grants to A.P.M [076566/Z/05/Z], J.C.K. [074318] and a core award to the Wellcome Trust Centre for Human Genetics [075491/Z/04].

1) Cell Culture
<ol>
<li>Select cell types expressing the gene of interest.</li>
<li>Select cell lines heterozygous both for DNA risk variants and transcribed coding polymorphism within the gene of interest (Figure 1).</li>
<li>Culture the selected cell lines according to the supplier's specifications and prepare 2 x 10 cm petri dishes for DNA and RNA extraction respectively.</li>
<li>When cells reach 80% confluence start the procedure to harvests cells and isolate DNA and RNA either following the procedure described below or using commercially available kits.</li>
</ol>
2) DNA Extraction
<ol>
<li>Remove media from the dish, wash with PBS and add to the dish 500 &mu;L of a lysis solution (0.6% SDS, 100 mM NaCl, 50 mM Tris, 20 mM EDTA and 50 &mu;g/mL RNase A).</li>
<li>Rock the plate gently for 20 minutes at room temperature.</li>
<li>Scrape the cell lysate into a microcentrifuge tube and incubate at 37 &deg;C overnight.</li>
<li>Add proteinase K to a final concentration of 100 &mu;g/mL and incubate at 37 &deg;C overnight.</li>
<li>Extract the DNA with an equal volume of phenol-chloroform pH 8, repeat, then extract with an equal volume of chloroform/isoamyl alcohol.</li>
<li>Precipitate the DNA with 1/10th volume 3 M NaCl and 2.5 volumes of 100% ethanol.</li>
<li>Leave on ice for 5 min and then spin at 13,000 rpm for 30 min at 4 &deg;C.</li>
<li>Wash the DNA with 70% ethanol, allow to air-dry and resuspend in 200 &mu;L of TE.</li>
<li>Incubate at 65 &deg;C for 10 min and then leave for 2 days at room temperature.</li>
<li>Store at 4 &deg;C or long term at -20 &deg;C.</li>
</ol>
3) RNA Extraction
<ol>
<li>Remove media from the dish, wash with PBS, add 1 mL of TRIzol and incubate for 10 min at room temperature.</li>
<li>Scrape cells, pipette a few times, transfer in a microcentrifuge tube and incubate for 5 min.</li>
<li>Spin for 10 min at 10,000 rpm at 4&deg;C.</li>
<li>Transfer the supernatant in a fresh tube and add 200 &mu;L of chloroform.</li>
<li>Shake and spin for 10 min at 10,000 rpm at 4&deg;C.</li>
<li>Transfer the aqueous phase in a fresh tube and add an equal volume of 70% ethanol.</li>
<li>Load the solution to 1 or 2 (depending on the volume) RNeasy column and spin for 30 sec at maximum speed.</li>
<li>From here follow instruction of the RNeasy kit (Qiagen).</li>
</ol>
4) Nucleic Acid Sample Preparation
<ol>
<li>Quantify DNA and RNA concentration using a NanoDrop Spectrophotometer (Thermo Scientific).</li>
<li>Prepare cDNA from 500-1000 ng of RNA using random decamers and SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions.</li>
</ol>
5) PCR and Primer Extension Assay
<ol>
<li>Design two pairs of PCR primers for each DNA marker, one pair for genomic DNA amplification and the other pair for cDNA amplification (Fig 2). Place forward and reverse primer for cDNA amplification in different exons to avoid genomic contamination. Design primer to obtain a PCR product in the range of 100-500 bp length.</li>
<li>Prepare a 10 &mu;L PCR reaction including 0.5 U Immolase Taq (Bioline) 0.8 mM dNTPs, 2 mM MgCl2, 0.2 M each primer and 20 ng of either cDNA or DNA. Amplify each sample in four independent reactions.</li>
<li>Run the PCR reaction under the conditions optimized for each primer pair keeping cycle number in linear phase of amplification.</li>
<li>Remove PCR primer and dNTPs excess by incubating the PCR product with exonuclease I and Shrimp Alkaline Phosphatase (SAP) at 37 &deg;C for 20 minutes.</li>
<li>Spot each PCR product twice on a 384-well plate, so that 8 measurements will be available for each sample.</li>
<li>Design the extension primer with the Assay Design (Sequenom) software so that the same primer can be used for both genomic and cDNA PCR products. Specify primer length in the range of 14-18 bp and extension mix including combinations of 3ddNTPs (Fig 2).</li>
<li>Carry out the primer extension reaction and MALDI-TOF/MS analysis using the MassARRAY system (Sequenom) according to the manufacturer's instruction.</li>
<li>A typical primer extension reaction include a starting step at 94 &deg;C for 2 min, followed by 40 cycles of 94 &deg;C for 5 s, 52 &deg;C for 5 s and 72 &deg;C for 5 s.</li>
<li>The primer extension products are cleaned with SpectroCLEAN resin and transferred onto microarray (chip) by SpectroPOINT nanolitre dispenser.</li>
<li>The extended oligonucleotides are quantified by MALDI-TOF using a SpectroREADER mass spectrometer.</li>
</ol>
6) Statistical Analysis
<ol>
<li>Check MALDI-TOF/MS results with the MassARRAY Typer software for general quality of the experiment.</li>
<li>Extract the Allelotyping report which includes the peak area data.</li>
<li>For each individual spectrum calculate the ratio between the peak area of allele 1 and allele 2 (peak area ratio).</li>
<li>For each cDNA sample derive a peak area ratio mean and standard deviation, using the corresponding function in Excel, across the 8 measurements.</li>
<li>Derive the same mean peak area ratio across the 8 measurments for genomic DNA.</li>
<li>Divide the cDNA mean ratio by the genomic mean ratio to assess the deviation to the expected 1:1 ratio.</li></ol>
7) Experimental Controls
<ol>
<li>To rule out experimental artifacts, repeat the experiment at least three times.</li>
<li>Whenever possible, design assays for additional DNA markers.</li>
<li>Design multiple PCR primer pairs and extension primers annealing to both forward and reverse strands.</li>
<li>Whenever possible test as negative controls cell lines that are heterozygous for DNA markers but do not carry the DNA risk variants associated with the phenotype (Fig. 1 and Fig. 3)</li>
</ol>
8) Representative Results
An example of allele-specific expression analysis is shown in Figure 3 with examples of mass spectrometry traces. The figure shows the results from the analysis of 4 allele-specific primer extension products carried out on 4 different templates. The top graphs represent the results obtained from the analysis of genomic DNA of two different cell lines, which are both heterozygous for a transcribed coding polymorphism. However, only cell line A is heterozygous for the risk DNA variant (Figure 1). The lower graphs represent the results obtained from the analysis of the cDNA generated from the same cell lines. As a result of experimental artifact the first peak is always higher than the second peak even in the genomic analysis. Therefore, the results from the genomic analysis are used to normalize the cDNA data. After normalization, the allele ratio is significantly different from 1 in cell line A (where the second peak appears lower compared to the other spectra), while it is very close to 1 in cell line B. The data suggest that cell line A carries a DNA variant, in phase with the allele measured by the second peak, which reduces the expression of the gene under analysis. Cell line B provides a very convenient negative control.
<img src="/files/ftp_upload/2279/2279fig1.jpg" alt="Figure 1" /> Figure 1. Cell line selection strategy. Cell lines A and B are heterozygous for a transcribed coding marker (triangle) but only cell line A is heterozygous for risk DNA variant (circle). The blue allele of the risk variant is associated (either with a direct effect or because in close correlation with the actual functional variant) with a lower level of transcription.
<img src="/files/ftp_upload/2279/2279fig2.jpg" alt="Figure 2" /> Figure 2. Allele-specific primer extension assay. This figure show the work-flow of the experimental procedure carried for both cDNA (left) and genomic DNA (right) templates. PCR primers (grey rectangles) are designed to amplify a heterozygous coding polymorphism (triangle). To avoid genomic contamination PCR primers anneal sequences placed in different exons (light blue bars) when amplification is carried out on cDNA template. The PCR product is then used as template for a primer extension reaction carried out with an extension primer, annealing one base next to the polymorphism, and the appropriate mix of three dideoxynucleotide (ddNTPs) terminators (squares) and one deoxynucleotide (circle) to extend the primer of one or two basis respectively. The resulting primer extension products have different masses which allow separation and quantification by MALDI-TOF mass spectrometry. The quantity of product corresponding to the blue allele of the DNA marker is relatively lower to the red allele.
<img src="/files/ftp_upload/2279/2279fig3.jpg" alt="Figure 3" /> 
 Figure 3. Results of an allele-specific expression assay. The figure show the mass spectrometry results from the analysis carried out on the genomic DNA and the cDNA of cell line A and cell line B (Figure 1).

<ol>
	<li>Cookson, W., Liang, L., Abecasis, G., Moffatt, M., Lathrop, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+complex+disease+traits+with+global+gene+expression.">Mapping complex disease traits with global gene expression.</a> Nat Rev Genet. 10, 184-194 (2009).</li><li>Singer-Sam, J., LeBon, J. M., Dai, A., Riggs, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive,+quantitative+assay+for+measurement+of+allele-specific+transcripts+differing+by+a+single+nucleotide.">A sensitive, quantitative assay for measurement of allele-specific transcripts differing by a single nucleotide.</a> PCR Methods Appl. 1, 160-163 (1992).</li><li>Yan, H., Yuan, W., Velculescu, V. E., Vogelstein, B., Kinzler, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelic+variation+in+human+gene+expression.">Allelic variation in human gene expression.</a> Science. 297, 1143-11 (2002).</li><li>Ding, C., Cantor, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+gene+expression+analysis+technique+using+competitive+PCR+and+matrix-assisted+laser+desorption+ionization+time-of-flight+MS.">A high-throughput gene expression analysis technique using competitive PCR and matrix-assisted laser desorption ionization time-of-flight MS.</a> Proc Natl Acad Sci U S A. 100, 3059-3064 (2003).</li><li>Gabriel, S., Ziaugra, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNP+genotyping+using+Sequenom+MassARRAY+7K+platform.">SNP genotyping using Sequenom MassARRAY 7K platform.</a> Curr Protoc Hum Genet Chapter. Chapter 2 (Unit 2), 12-12 (2004).</li><li>Lo, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelic+variation+in+gene+expression+is+common+in+the+human+genome.">Allelic variation in gene expression is common in the human genome.</a> Genome Res. 13 (8), 1855-1862 (2003).</li><li>Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression-based+discovery+of+variation+in+the+human+glutathione+S-transferase+M3+promoter+and+functional+analysis+in+a+glioma+cell+line+using+allele-specific+chromatin+immunoprecipitation.">Expression-based discovery of variation in the human glutathione S-transferase M3 promoter and functional analysis in a glioma cell line using allele-specific chromatin immunoprecipitation.</a> Cancer Res. 65, 99-104 (2005).</li><li>Knight, J. C., Keating, B. J., Rockett, K. A., Kwiatkowski, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+characterization+of+regulatory+polymorphisms+by+allele-specific+quantification+of+RNA+polymerase+loading.">In vivo characterization of regulatory polymorphisms by allele-specific quantification of RNA polymerase loading.</a> Nat Genet. 33 (4), 469-475 (2003).</li><li>Pastinen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+genetic+and+epigenetic+variation+affecting+human+gene+expression.">A survey of genetic and epigenetic variation affecting human gene expression.</a> Physiol Genomics. 16 (2), 184-193 (2004).</li><li>Francks, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+77-kilobase+region+of+chromosome+6p22.2+is+associated+with+dyslexia+in+families+from+the+United+Kingdom+and+from+the+United+States.">A 77-kilobase region of chromosome 6p22.2 is associated with dyslexia in families from the United Kingdom and from the United States.</a> Am J Hum Genet. 75 (6), 1046-1058 (2004).</li><li>Paracchini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chromosome+6p22+haplotype+associated+with+dyslexia+reduces+the+expression+of+KIAA0319,+a+novel+gene+involved+in+neuronal+migration.">The chromosome 6p22 haplotype associated with dyslexia reduces the expression of KIAA0319, a novel gene involved in neuronal migration.</a> Hum Mol Genet. 15 (10), 1659-1666 (2006).</li></ol>

No conflicts of interest declared.

This method enables evaluation of the effect of disease-associated DNA variants on gene expression by assessing in vivo allele-specific difference in transcript level. In this protocol relative transcript abundance is measured by MALDI-TOF but other technologies allowing allele-specific quantification, such as TaqMan6,7, can be used. The major limitation of this approach is the availability of transcribed coding markers. Methods based on the principle described here, but taking advantage of different classes of polymorphisms, have been described. The haploChip assay measures allele-specific expression using markers located within 1 kb of the transcriptional start or end site of a gene which would be present in chromatin immunoprecipitated material isolated with antibodies specific to RNA polymerase II8. Alternatively, intronic SNPs can be used when the template is heteronuclear RNA9.
The specific protocol described here, in combination with the haploChip assay, has been used successfully to identify a candidate gene for dyslexia (or reading disability). Initially a genetic association study identified a DNA sequence associated with dyslexia and which was spanning three genes10. The allele-specific gene expression assay showed that in the presence of the dyslexia-associated DNA variants expression variation was observed for only one of the three genes but not the other two11.

<table><tbody><tr><td>PBS</td><td>Sigma-Aldrich</td><td>P-4417-100TAB</td><td></td></tr><tr><td>SDS</td><td>Bio-Rad</td><td>161-0416</td><td></td></tr><tr><td>NaCl</td><td>VWR international</td><td>27788.366</td><td></td></tr><tr><td>Tris</td><td>Sigma-Aldrich</td><td>T1503-1KG</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E5134-500G</td><td></td></tr><tr><td>RNase A</td><td>Qiagen</td><td>19101</td><td></td></tr><tr><td>Proteinase K</td><td>Sigma-Aldrich</td><td>P2308-500MG</td><td></td></tr><tr><td>Phenol: chloroform</td><td>Sigma-Aldrich</td><td>77617</td><td></td></tr><tr><td>Chloroform</td><td>VWR international</td><td>100777C</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>32221-2.5L</td><td></td></tr><tr><td>Trizol</td><td>Invitrogen</td><td>15596-018</td><td></td></tr><tr><td>RNeasy kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>SuperScript III Reverse Transcriptase</td><td>Invitrogen</td><td>18080-044</td><td></td></tr><tr><td>Immolase Taq</td><td>Bioline</td><td>BIO-21048</td><td></td></tr><tr><td>dNTP</td><td>Sigma-Aldrich</td><td>DNTP100A</td><td></td></tr><tr><td>Exonuclease 1</td><td>New England Biolabs</td><td>M0293S</td><td></td></tr><tr><td>Shrimp Alkaline Phosphatase</td><td>GE Healthcare</td><td>E70092Z</td><td></td></tr><tr><td>SpectroCLEAN resin</td><td>Sequenom</td><td>10053</td><td></td></tr><tr><td>MassEXTEND ddNTP/dNTP mix </td><td>Sequenom</td><td></td><td>Varies according to assay design</td></tr><tr><td>MassEXTEND thermosequenase </td><td>Sequenom</td><td>10052</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Chilled microcentrifuge</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>NanoDrop Spectrophotometer</td><td>Thermo Fisher Scientific, Inc.</td><td></td><td></td></tr><tr><td>SpectroPOINT nanolitre dispenser</td><td>Sequenom</td><td></td><td></td></tr><tr><td>SpectroREADER mass spectrometer</td><td>Sequenom</td><td></td><td></td></tr></tbody></table>

an allele-specific gene expression assay to test the functional basis of genetic associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Watch this Scientific Journal Video about An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations at JoVE.com

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

an-allele-specific-gene-expression-assay-to-test-functional-basis

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

22.8K Views.  University of Oxford. Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

Video: An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

Genetics

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3&#39; region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

In the present study, we describe a methodology to analyze the C924T genotype. The protocol consists of three phases: DNA extraction, amplification by polymerase chain reaction (PCR), and analysis of the restriction fragment length polymorphism (RFLP) on agarose gel.

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

Determining the Likelihood of Variant Pathogenicity Using Amino Acid-level Signal-to-Noise Analysis of Genetic Variation

Advancements in the cost and speed of next generation genetic sequencing have generated an explosion of clinical whole exome and whole genome testing. While this has led to increased identification of likely pathogenic mutations associated with genetic syndromes, it has also dramatically increased the number of incidentally found genetic variants of unknown significance (VUS). Determining the clinical significance of these variants is a major challenge for both scientists and clinicians. An approach to assist in determining the likelihood of pathogenicity is signal-to-noise analysis at the protein sequence level. This protocol describes a method for amino acid-level signal-to-noise analysis that leverages variant frequency at each amino acid position of the protein with known protein topology to identify areas of the primary sequence with elevated likelihood of pathologic variation (relative to population &#34;background&#34; variation). This method can identify amino acid residue location &#34;hotspots&#34; of high pathologic signal, which can be used to refine the diagnostic weight of VUSs such as those identified by next generation genetic testing.

Amino acid-level signal-to-noise analysis determines the prevalence of genetic variation at a given amino acid position normalized to background genetic variation of a given population. This allows for identification of variant &#34;hotspots&#34; within a protein sequence (signal) that rises above the frequency of rare variants found in a population (noise).

Advancements in the cost and speed of next generation genetic sequencing have generated an explosion of clinical whole exome and whole genome testing. ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Cancer Research

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

Medicine

Large-Scale Multi-Omics Genome-Wide Association Studies (Mo-GWAS): Guidelines for Sample Preparation and Normalization

Both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) are widely used metabolomics approaches to detect and quantify hundreds of thousands of metabolite features. However, the application of these techniques to a large number of samples is subject to more complex interactions, particularly for genome-wide association studies (GWAS). This protocol describes an optimized metabolic workflow, which combines an efficient and fast sample preparation with the analysis of a large number of samples for legume crop species. This slightly modified extraction method was initially developed for the analysis of plant and animal tissues and is based on extraction in methyl tert-butyl ether: methanol solvent to allow the capture of polar and lipid metabolites. In addition, we provide a step-by-step guide for reducing analytical variations, which are essential for the high-throughput evaluation of metabolic variance in GWAS.

Large-Scale Multi-Omics Genome-Wide Association Studies Mo-GWAS: Guidelines for Sample Preparation and Normalization

In this protocol, we present an optimized workflow, which combines an efficient and fast sample preparation of many samples. In addition, we provide a step-by-step guide to reduce analytical variations for high-throughput evaluation of metabolic GWAS studies.

Both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) are widely used metabolomics approaches to detect ...

Biochemistry

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.