The authors have no acknowledgements.

The genetic material used herein was ethically approved for use by the Office for Children HREC (Human Research Ethics Committee) (CDF/07/492), the Department of Human Services HREC (10/07) and the Royal Children&#8217;s Hospital (RCH) HREC (27047).
1. Designing the Multiplexed Assay
<ol>
	<li>Prepare a SNP list for assay design.
	<ol>
		<li>Input the target region into the tagger function of Haploview (https://www.broadinstitute.org/haploview/downloads). Use the LD (correlation, r2) between SNPs spanning the target region to generate a list of SNPs, which provides full coverage of the desired region with the fewest number of required variants. Generate a list of SNPs such that all alleles to be captured are correlated above a user-defined threshold (e.g., r2&#160;&#8805;0.8).</li>
	</ol>
	</li>
	<li>Identification of suitable sequences for forward, reverse, and extension primers
	<ol>
		<li>Enter the generated list of SNPs into the assay design tool of choice. 
		Note: The following instructions apply to the assay design tool used to generate the representative results, as specified in the Table of Materials.</li>
		<li>Once the assay design tool has been launched, select New Genotyping Design. Enter a name for the assay design into the Design Name field.</li>
		<li>Provide the SNP list generated in step 1.1.1 by clicking on the button labeled Edit Text Input with the instructions rs or FASTA to the left of the button. 
		Note: SNPs can be entered as a text list of reference (rs) SNP IDs or uploaded in FASTA format, or the SNP Group file format used by the tool. To upload files in these formats, select the File Upload button. The upper limit of variants that can be designed into a single assay, termed &#8216;well,&#8217;&#160;is 40 SNPs. It is possible to specify SNPs that are a priority for the design before starting the run; these will be designed first. Control SNPs can be specified: for example, an assay to detect gender in case of sample mix ups or a control to determine that sufficient template was added if working with low or poor quality DNA. However, it should be noted that using control and priority SNPs can reduce the number of SNPs that can be designed into a single well.</li>
		<li>From the Preset menu, select the High Multiplexing iPLEX Preset option. From the Organism menu, select the organism from which the DNA to be genotyped derives. For the representative results presented here, this was Human. 
		Note: Mouse and bovine organisms are also compatible with the tool. One can also select Other if working with an alternative organism such as plant or microbe; however, the automated steps for finding proximal SNPs and optimal primer areas will not be available due to the absence of a reference genome.</li>
		<li>At the Database menu, select the latest build of the genome, or an alternative build if required, from the Chemistry menu. Select iPLEX and for the Multiplex Level field, enter the highest level of multiplexing: 40. Now, select Begin Run. &#8220;Step 1&#8221; of the tool will commence. 
		Note: During step 1, the tool retrieves and formats the SNP sequences. Here, errors might include formatting of the FASTA file in which case the FASTA file should be checked and reformatted as necessary. Another error is SNP rs numbers that have been merged with another rs number, in which case the rs number should be searched in a SNP database such as the NCBI&#8217;s dbSNP to identify the new SNP rs number. Sequences around the SNP are searched for any known proximal SNPs that could interfere with the primer design. Potential PCR primer sites are compared against the genome to avoid non-specific amplification due to multiple hits in the genome.</li>
		<li>Await completion of &#8220;Step 2&#8221; of the design tool in which proximal SNPs will be identified. 
		Note: If using an organism that is not supported by this step, annotate the sequences with proximal SNPs in IUPAC annotation if this information is available. There are rarely errors during this step; however, errors may occur if the wrong reference organism or genome was selected, or if cDNA sequences were entered rather than genomic DNA. To optimize step 2, under Advanced Settings, it is possible to filter out SNPs based on a population if working within a specific population. Rare SNPs can also be filtered out by excluding those with a frequency below 1%. Finally, SNPs that do not have a validated status or do not have population information can also be excluded if desired.</li>
		<li>Await completion of &#8220;Step 3&#8221; of the design tool that identifies optimal primer regions. If preferred primer locations have been identified, or if working with a different organism, manually input primer locations by annotating the input sequence (the SNP group files) with the preferred primer regions in uppercase and the rest of the sequence in lower case, select Preserve Case from the Advanced Settings menu for this step. 
		Note: Errors for Steps 2 &#38; 3 may include: (1) A proximal SNP blocking the design of a primer with the solution being to mask with a non-template base and order an oligo with a universal base like Isonine, or, design two primers, one with each of the alleles of the proximal SNP, or, design one primer with the common allele only. (2) Another common error is the failure to identify a unique site for the primer with the solution being to modify the amplicon length in order to identify a unique site. In the tool used for this paper, the default is 80 - 120 bp. This can be altered from 60 - 400 bp in the advanced settings dialogue under the Amplicon Length settings in &#8220;3. Identify Optimal Primer Areas&#8221; and also under the Amplicon tab of &#8220;4. Design Assays.&#8221;&#160;Ensure both are changed. However, it should be noted that increasing the length of the amplicon when using degraded DNA is not recommended.</li>
		<li>Await completion of the assay design step (&#8220;step 4&#8221; of the design tool), here the tool aims to provide optimal pass separation between the extension primers and the alleles of all the SNPs in the multiplexed designs. After this step is complete, export the Oligo Order file, which is formatted for easy ordering through an oligo ordering vendor of choice. 
		Note: Amplification primers (Forward (F) and Reverse (R)) are designed to produce an optimal amplicon size of 80 to 120 bp. The amplification primer mass must differ from that of the extension primer and its product to avoid conflicting results in the mass spectrum and for overall PCR performance. The extension primer should be 15 to 30 nucleotides long (4,500 - 9,000 Da), designed so that the 3&#8217; end will attach directly adjacent to the site of the polymorphism. All of these settings are automatically set in the tool used to generate the representative results.</li>
	</ol>
	</li>
	<li>Export the design from the chosen tool and order primers from an oligomer provider: 25 nmol of each of the PCR primers (forward and reverse), and 100 nmol of each of the extension primers. See Table 1 for the primers used to generate the representative results.</li>
</ol>
2. Genotyping (1 - 2 days)
<ol>
	<li>Prepare amplification primers.
	<ol>
		<li>If lyophilized primers were ordered, reconstitute with molecular grade water. Use a concentration of 100 &#956;M/&#956;L for forward and reverse primers and 500 &#956;M/&#956;L for extension.</li>
		<li>Pool forward &#38; reverse primers for each multiplexed assay into a stock primer mix, containing each primer at a concentration of 0.5 mM. 
		Note: For example, a 400 &#956;L primer pool for 400 reactions: 
		<img alt="Equation" src="/files/ftp_upload/57601/57601eq1v2.jpg" /> 
		2 &#956;L of each primer, at a concentration of 100 &#956;M/&#956;L, is required for the stock primer pool. For a 30-plex assay with 60 forward and reverse primers, a total of 120 &#956;L of primer would be required (the concentrated primer pool). 280 &#956;L molecular-grade water would then be added to make a final volume of 400 &#956;L of primer mix.</li>
	</ol>
	</li>
	<li>Amplification using Polymerase Chain Reaction (PCR)
	<ol>
		<li>Make a master mix for the PCR containing 100 nM of the primer mix detailed above, 500 &#956;M of dNTPs, 2 mM of MgCl2, and 0.5 U of a DNA polymerase enzyme. 1 U is required for greater than 26-plex assays. Refer to Table 2.</li>
		<li>Add 4 &#956;L of this mix to each well of the 384-well PCR plate, along with 1 &#956;L of genomic DNA at a concentration of 5 - 10 ng/&#956;L. 
		Note: Quality controls should also be included on the plate, such as a non-template control, and a positive control that has previously performed well with the assay (during a test run). If running multiple plates, multiple DNA samples from the other plate(s), e.g., in triplicate, should be run as technical controls for inter-plate variability.</li>
		<li>Centrifuge the plate at 200 x g for 1 min at room temperature with a plate cover in place to ensure all liquid is contained at the bottom of the plate wells.</li>
		<li>Run the PCR with the following thermocycler conditions: 4 min incubation at 94 &#176;C to activate the DNA polymerase; 45 cycles of the following (denaturation at 94 &#176;C for 20 s, annealing at 56 &#176;C for 30 s, and extension at 72 &#176;C for 1 min) and then a final extension at 72 &#176;C for 3 min. Refer to PCR program in Table 3.</li>
		<li>Perform agarose gel electrophoresis to ensure the DNA amplification is successful. Use 1 &#181;L of PCR product (with 3 &#181;L of loading buffer) on a 2% (w/v) agarose gel, made by mixing agarose powder and 0.5% Tris Borate EDTA (TBE) buffer, and run for 45 min at 100 V.</li>
	</ol>
	</li>
	<li>Shrimp Alkaline Phosphatase (SAP) Purification Step 
	Note: This step is used to remove unincorporated nucleotides. The SAP enzyme cleaves phosphate groups to prevent excess primer and unincorporated dNTPs from interfering in the upcoming extension reaction.
	<ol>
		<li>Make a master mix containing 1.7 units/&#956;L of Shrimp Alkaline Phosphatase (SAP) enzyme, 10x SAP buffer and make up to volume with molecular-grade water. Refer to Table 4.</li>
		<li>Add 2 &#956;L of the freshly made up SAP master mix to each well of the plate, already containing 5 &#956;L from the PCR reaction. Centrifuge briefly and return to thermocycler at 37 &#176;C for 40 min and then at 85 &#176;C for 5 min for enzyme inactivation. Refer to Table 5.</li>
	</ol>
	</li>
	<li>Primer Extension Reaction 
	Note: The concentration of the extension primers in the extension primer mix must be adjusted by size (Linear Primer Adjustment &#8211; LPA). This is because there is an inverse relationship in peak intensity (on the mass spectra) and product mass. Adjustment of the extension primer concentration corrects for this relationship to produce peaks of equal intensities. Adjusted primers used to generate the representative results are provided in Tables 6 &#38; 7. The volumes provided are to generate 1.5 mL of extension primer mix.
	<ol>
		<li>Calculate dilution factors for each primer using a gradient algorithm, available from Agena Biosciences (&#8216;Linear Primer Adjustment&#8217;), which adjusts for the mass and concentration of the primer, and the total number of primers in the multiplexed reaction (see Tables 6 &#38; 7). Enter the UEP mass for each primer into the UEP_MASS field of the Linear Primer Adjustment sheet. 
		Note: The mass adjusted concentration for the primer will be automatically calculated in cell with the header text &#8220;Target Reaction Concentration (&#181;M).&#8221;&#160;The volume of primer to be added to the primer pool will be output in the cell under the header text &#8220;Volume Stock Primer to be Added to Pool (&#181;L).&#8221;&#160;The volume of water to be added to the primer pool to make up the calculated concentrations is output to the cell next to the text &#8220;Volume of PCR Grade RNase free H2O to be added Primer Pool (&#181;L).&#8221;</li>
		<li>Take the volume of each primer as specified in the Linear Primer Adjustment file and make up a primer pool. Add the volume of water calculated by the algorithm to dilute the primer pool as determined in step 2.4.1. 
		Note: If the gradient algorithm method is unavailable, another method is to divide the primers into low mass and high mass and add double the concentration of the high mass primers relative to the low mass group. Three or four groups may be required for a high-plex assay (that is, more than 19 SNPs). However, this method will not yield as optimal call rates as the gradient algorithm method.</li>
		<li>Assemble the extension master mix. For the low plex assay (1 - 18 plex), add 0.2 &#956;L of buffer, 0.1 &#956;L of termination mix, 0.94 &#956;L of the LPA adjusted primer mix, and 0.0205 &#956;L of enzyme (prepared on ice). For the high plex assay (19 - 36 plex), add double concentration of termination mix and enzyme (termination mix 0.2 &#956;L and iPLEX enzyme 0.041 &#956;L). Refer to Table 8.</li>
		<li>Add 2 &#956;L of the extension reaction master mix to the plate from the previous reaction, now bringing the total volume to 9 &#956;L per well. Centrifuge briefly and place in the thermocycler: 94 &#176;C for a 30 s initial incubation, 40 cycles of 1 x 94 &#176;C for 5 s with 5 x (52 &#176;C for 5 s, followed by 80 &#176;C for 5 s), and then 72 &#176;C for 3 min. Refer to Table 9.</li>
	</ol>
	</li>
	<li>Resin purification. 
	Note: Excess salts can now be removed from the reaction with the use of resin.
	<ol>
		<li>Add 16 &#956;L of deionized water to the wells of the retrieved plate, bringing the volume in the plate up to 25 &#956;L.</li>
		<li>Apply resin, usually 6 mg of dimple resin plate (purchased separately), evenly to the whole plate. Leave the resin to dry in the resin dimple plate for 20 min at room temperature before applying to the reaction plate. After addition of the resin, rotate plate for 5 min at room temperature either by hand or with a suspension mixer at a slow speed (e.g., 7.5 rpm). 
		Note: Prior to loading the genotyping analyte onto the genotyping chip, the samples and the assay layout must be input into the software interface. This can be achieved using the Design Summary file from the assay design tool.</li>
	</ol>
	</li>
	<li>Measurement on a mass spectrometry platform.
	<ol>
		<li>Centrifuge the plate at 3,200 x g for 5 min to avoid application of resin to the mass spectra chip.</li>
		<li>Upload the plate layout of samples to the system&#8217;s software interface prior to the run. 
		Note: Step 2.6.3 should only be performed by trained personnel.</li>
		<li>Apply the genotyping analyte mixture from the plate to the genotyping chip with a dispensing system. Next, load the chip onto the MALDI-TOF system to generate the mass spectra from the analyte mixture, which can then be interpreted with the aid of the platform&#8217;s software interface. 
		Note: The system generates the mass spectra by directing short pulses of UV light at each &#8216;pad,&#8217;&#160;corresponding to a single well of the genotyping plate, of the mass spectra chip. This pulse results in desorption and ionization of the analyte. These ionized DNA molecules are then propelled to the top of the vacuum tube by a high voltage electrostatic field, separating out lighter ions, which travel faster and reach the detector first, from the heavier ions. The &#8220;time of flight&#8221; of these analytes are recorded by the system, from which the mass of each DNA fragment can be determined and thus the nucleotide base present at the SNP can be inferred.</li>
	</ol>
	</li>
</ol>
3. Genotyping Data
Note: Quality control and data interpretation are not the focus of this methodology paper. However, the following will briefly cover interpretation of the mass spectra.
<ol>
	<li>Manual inspection and quality control of mass spectra. 
	Note: The raw mass spectra are generated by the system and analyzed with the software as listed in the Table of Materials. A Gaussian mixture clustering method is applied to this output to make probabilistic genotyping calls.
	<ol>
		<li>Open the analysis software. Select Typer Analyzer. From the File menu, select Open Wells from File and select the xml file with the spectrum data generated in step 2.6.3. The chip name for the run should be visible, select it and a &#8220;traffic light&#8221; pane of the 384-well plate will be displayed with a list of the SNPs in the selected well. From here, select Call Cluster Plots to view a scatterplot of data points for each SNP. 
		Note: It is recommended that a &#8216;no call&#8217; result be applied for low intensity SNPs. In the software used for the representative results, a clustering magnitude cut-off of 5, which is higher than default, is suggested for stringent quality control. This setting can be adjusted in the Magnitude Cutoff field under Parameters in the Post Processing Clusters pane. Select Autocluster from the Tools tab to run a cluster analysis.</li>
		<li>Perform manual inspection of genotype calls by examining the Cartesian Plots in the Post Processing Pane. In the Assay pane, select the SNP of interest and click on the Post-Processing Clusters tab, a cluster plot with genotype clustering will be visible with sample IDs and corresponding genotypes for the SNP.</li>
		<li>Examine the genotype call clusters and assess how well each individual call clusters with the other calls for the SNP. The software provides some boundary lines on the plot for each genotype for guidance; see example of a genotype calls cluster plot from an IL13 SNP (Figure 1). If the call does not cluster tightly with other calls and sits clearly outside of a cluster, or if it has very low intensity, right click the datapoint and select Change Call to manually set it to No Call.</li>
		<li>To ensure high quality genotyping data is used for downstream analysis, exclude SNPs and individuals with call rates below a QC threshold, e.g. 90%. Once appropriate adjustments have been made to the call data, export the data for statistical applications. In the software used to generate the representative results (listed in the Table of Materials) this is achieved with the Plate Data selection from the view menu and selecting Save As.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Aronson, S. J., Rehm, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+the+foundation+for+genomics+in+precision+medicine.">Building the foundation for genomics in precision medicine.</a> Nature. 526 (7573), 336-342 (2015).</li><li>Ball, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+a+GWAS:+power,+sample+size,+and+data+structure.">Designing a GWAS: power, sample size, and data structure.</a> Genome-Wide Association Studies and Genomic Prediction. , 37-98 (2013).</li><li>Kwon, J. M., Goate, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+candidate+gene+approach.">The candidate gene approach.</a> Alcohol research and health. 24 (3), 164-168 (2000).</li><li>Oeth, P., Mistro, G. D., Marnellos, G., Shi, T., van den Boom, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualitative+and+quantitative+genotyping+using+single+base+primer+extension+coupled+with+matrix-assisted+laser+desorption/ionization+time-of-flight+mass+spectrometry+(MassARRAY).">Qualitative and quantitative genotyping using single base primer extension coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MassARRAY).</a> Single Nucleotide Polymorphisms: Methods and Protocols. , 307-343 (2009).</li><li>Pusch, W., Kostrzewa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+MALDI-TOF+mass+spectrometry+in+screening+and+diagnostic+research.">Application of MALDI-TOF mass spectrometry in screening and diagnostic research.</a> Current pharmaceutical design. 11 (20), 2577-2591 (2005).</li><li>Su, K. Y., 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=Pretreatment+epidermal+growth+factor+receptor+(EGFR)+T790M+mutation+predicts+shorter+EGFR+tyrosine+kinase+inhibitor+response+duration+in+patients+with+non-small-cell+lung+cancer.">Pretreatment epidermal growth factor receptor (EGFR) T790M mutation predicts shorter EGFR tyrosine kinase inhibitor response duration in patients with non-small-cell lung cancer.</a> Journal of clinical oncology. 30 (4), 433-440 (2012).</li><li>Singhal, N., Kumar, M., Kanaujia, P. K., Virdi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI-TOF+mass+spectrometry:+an+emerging+technology+for+microbial+identification+and+diagnosis.">MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis.</a> Frontiers in microbiology. 6, 791 (2015).</li><li>Cricca, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+genotyping+of+high-risk+Human+Papillomavirus+by+MALDI-TOF+Mass+Spectrometry-based+method.">High-throughput genotyping of high-risk Human Papillomavirus by MALDI-TOF Mass Spectrometry-based method.</a> New Microbiologica. 38 (2), 211-223 (2015).</li><li>Ashley, S., 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=Genetic+Variation+at+the+Th2+Immune+Gene+IL13+is+Associated+with+IgE-mediated+Paediatric+Food+Allergy.">Genetic Variation at the Th2 Immune Gene IL13 is Associated with IgE-mediated Paediatric Food Allergy.</a> Clinical &amp; Experimental Allergy. 47 (8), 1032-1037 (2017).</li><li>Bousman, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+NRG1+and+DAOA+genetic+variation+on+transition+to+psychosis+in+individuals+at+ultra-high+risk+for+psychosis.">Effects of NRG1 and DAOA genetic variation on transition to psychosis in individuals at ultra-high risk for psychosis.</a> Translational psychiatry. 3 (4), e251 (2013).</li><li>Moffatt, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large-scale,+consortium-based+genomewide+association+study+of+asthma.">A large-scale, consortium-based genomewide association study of asthma.</a> New England Journal of Medicine. 363 (13), 1211-1221 (2010).</li><li>Granada, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+association+study+of+plasma+total+IgE+concentrations+in+the+Framingham+Heart+Study.">A genome-wide association study of plasma total IgE concentrations in the Framingham Heart Study.</a> Journal of Allergy and Clinical Immunology. 129 (3), 840-845 (2012).</li><li>Oeth, P., 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=iPLEX+assay:+Increased+plexing+efficiency+and+flexibility+for+MassARRAY+system+through+single+base+primer+extension+with+mass-modified+terminators.">iPLEX assay: Increased plexing efficiency and flexibility for MassARRAY system through single base primer extension with mass-modified terminators.</a> Sequenom application note. 27, (2005).</li><li>Ellis, J. A., Ong, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MassARRAY+System+for+Targeted+SNP+Genotyping.">The MassARRAY System for Targeted SNP Genotyping.</a> Genotyping: Methods and Protocols. , 77-94 (2017).</li><li>Johnson, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNAP:+A+web-based+tool+for+identification+and+annotation+of+proxy+SNPs+using+HapMap.">SNAP: A web-based tool for identification and annotation of proxy SNPs using HapMap.</a> Bioinformatics. 24 (24), 2938-2939 (2008).</li></ol>

The authors have nothing to disclose.

Here, we demonstrate the method of multiplexed genotyping using mass spectrometry. The representative results were generated using PCR paired with MALDI-TOF mass spectrometry4 with assay chemistry listed in the Table of Materials13. With this platform, we generated a total of 11,295 genotypes on 1,255 individuals for 9 SNPs within 40 h in the lab.
We illustrate the usefulness of the technique in answering genetic hypotheses by ge...

In human complex disease, genetic variants contribute to disease susceptibility and quantifying these variants may be useful for understanding pathogenesis, identifying high risk patient groups, and treatment responders. Indeed, the promise of precision medicine is dependent upon utilizing genomic information to identify patient subgroups1. Unfortunately, within the complex disease biology space, where disease phenotypes are underpinned by substantial genetic heterogeneity, low penetrance, and variable expressivity, cohort size requirements for genome-wide approaches to identify novel candidates are often prohibitively large2. Alternatively, a targeted candidate gene approach begins with an a priori hypothesis about specific genes/pathways in disease etiology3. Pathway analysis tools are commonly used to investigate the pathophysiology of an identified target loci, generating numerous candidate pathways to be explored. We demonstrate here a multiplexed genotyping approach allowing for the investigation of tens to hundreds of SNPs with one assay, suited to human cohort studies4. This approach is relatively high through-put, permitting hundreds to thousands of DNA samples to be genotyped for novel discovery studies and investigation of specific pathways. The methods outlined here are useful for identifying risk alleles and their associations with clinical traits in a relatively rapid and inexpensive manner. This platform has been highly advantageous for screening and diagnostic purposes5,6, and more recently, for microbial infection7 and human papillomavirus8.
This protocol begins with selection of a set of genes for investigation, i.e., the target regions, typically determined through literature searching, or a priori hypotheses for involvement in the disease process; or perhaps selected for replication as the leading associations of a discovery genome-wide association (GWA) study. From the gene set, the researcher will select a refined list of tag SNPs. That is, the linkage disequilibrium (LD), or correlation, amongst variants in the region is used to identify a representative &#39;tag SNP&#39; for a group of SNPs in high LD, known as a haplotype. The high LD of the region means that the SNPs are often inherited together such that genotyping one SNP is sufficient to represent the variation at all SNPs in the haplotype. Alternatively, if following up on a definitive list of SNPs from many regions, e.g., replication for a GWA study, this process may be unnecessary. For multiplexed genotyping, an assay must then be designed around these targets such that the amplification primers are of differing mass to those of the extension primers and products to produce interpretable mass spectra. These parameters are easily implemented by a multiplexed genotyping assay design tool. The forward and reverse primers from this design will be used to target the markers of interest and amplify the sequence containing the SNP. The extension primers attach directly proximal to the SNPs and a single, mass-modified, &#39;terminator&#39; base that is complementary to the SNP is added. The terminator base prevents further extension of the DNA. The mass-modification of the base enables fragments differing by a single base to be detected by mass spectrometry. The plate containing the genotyping chemistry is then applied to a chip for measurement on a mass spectrometry platform. After applying appropriate quality controls to the raw genotyping calls detected by the system, the data can be exported and used for statistical analysis to test for association with disease phenotypes.

<table border="0" cellpadding="0" cellspacing="0" width="695">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Genomic DNA&#160;</td>
			<td style="width:127px;">-</td>
			<td style="width:119px;">-</td>
			<td style="width:235px;">1 &#956;L at a concentration of 5-10 ng/&#956;L</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Primers: forward and reverse amplification and extension</td>
			<td style="width:127px;">IDT</td>
			<td style="width:119px;">-</td>
			<td style="width:235px;">see manuscript section 1.2.1 on design of primers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deionized water&#160;</td>
			<td style="width:127px;">E.g. Milli-Q water&#160;</td>
			<td style="width:119px;">-</td>
			<td>deionized with 18.2 M&#937;.cm resistivity</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Genotyping reagent kit. iPLEX Gold Chemistry reagent set&#160;</td>
			<td>Agena Bioscience</td>
			<td style="width:119px;">#10148-2</td>
			<td style="width:235px;">includes all reagents for reactions in 2.2.1, 2.3.1 and 2.4.2 , chip and resin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">PCR plates (384-well)</td>
			<td style="width:127px;">Abgene</td>
			<td style="width:119px;">#ABGAB-1384</td>
			<td style="width:235px;">For the MassARRAY system plates by Abgene are compatible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipettes</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td>single and 8-channel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge&#160;</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td>compatible with 384-well plates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Thermocycler</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:235px;">compatible with PCR programs as detailed in 2.2.4, 2.3.2 and 2.4.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimple resin plate&#160;</td>
			<td>Agena Bioscience</td>
			<td style="width:119px;"></td>
			<td>6mg, 384-well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate rotator&#160;</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">MassARRAY Analyzer 4 System</td>
			<td style="width:127px;">Agena Biosciences</td>
			<td style="width:119px;"></td>
			<td style="width:235px;">MALDI-TOF (matrix-assisted laser desorption/ionization &#8211; time of flight) Mass Spectrometer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RS1000 Nanodispenser</td>
			<td style="width:127px;">Agena Biosciences</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Assay Design Suite</td>
			<td style="width:127px;">Agena Biosciences</td>
			<td style="width:119px;"></td>
			<td>Tool used to design the multiplex genotyping assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hot Start Taq</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td>DNA polymerase enzyme&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Resin&#160;</td>
			<td style="width:127px;">Agena Biosciences</td>
			<td style="width:119px;"></td>
			<td>Supplied with iPLEX kit</td>
		</tr>
	</tbody>
</table>

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.

With the protocol described above, we genotyped tag SNPs across the Th2 immune gene IL13 in a cohort of food allergy cases and controls9. We applied logistic regression analysis, adjusted for ancestry and other potential covariates, to test whether the genetic variants within the region of interest increased food allergy risk. Table 109 shows that one variant rs1295686 is associated with challenge-proven food allerg...

Watch this Scientific Journal Video about Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry at JoVE.com

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

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 ...

candidate-gene-testing-clinical-cohort-studies-with-multiplexed

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

10.1K Views.  Max-Delbrück Center for Molecular Medicine. 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.

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

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics (MSPP)

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. Allelic isoforms of the detectable biomarker ions result in isolate-specific mass shifts. Mass spectrometry-based phyloproteomics (MSPP) is a novel technique that combines the mass spectrometric detectable biomarker masses in a scheme that allows deduction of phyloproteomic relations from isolate specific mass shifts compared to a genome sequenced reference strain. The deduced amino acid sequences are then used to calculate MSPP-based dendrograms.
Here we describe the workflow of MSPP by typing a Campylobacter jejuni ssp. doylei isolate collection of seven strains. All seven strains were of human origin and multilocus sequence typing (MLST) demonstrated their genetic diversity. MSPP-typing resulted in seven different MSPP sequence types, sufficiently reflecting their phylogenetic relations.
The C. jejuni ssp. doylei MSPP scheme includes 14 different biomarker ions, mostly ribosomal proteins in the mass range of 2 to 11 kDa. MSPP can in principle, be adapted to other mass spectrometric platforms with an extended mass range. Therefore, this technique has the potential to become a useful tool for strain level microbial typing.

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics MSPP

Mass spectrometry-based phyloproteomics (MSPP) was used to type a collection of Campylobacter jejuni ssp. doylei isolates at the strain level in comparison to multilocus sequence typing (MLST).

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. ...

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is described. DNA extraction is carried out using a silica membrane-based DNA extraction kit. After the PCR amplification using a detergent-free PCR buffer, restriction enzymes are added to the solution without purifying the reaction solution. A reverse-phase silica monolith column and a Fourier transform high resolution mass spectrometer having a modified Kingdon trap analyzer are employed for separation and detection, respectively. The mobile phase, consisting of 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 15 mM triethylamine (pH 7.9) and methanol, is delivered at a flow rate of 0.4 ml/min. The cycle time for LC/ESI-MS analysis is 8 min including equilibration of the column. Deconvolution software having an isotope distribution model of the oligonucleotide is used to calculate the corresponding monoisotopic mass from the mass spectrum. For analysis of oligonucleotides (range 26-79 nucleotides), mass accuracy was 0.62 &#177; 0.74 ppm (n = 280) and excellent accuracy and precision were sustained for 180 hr without use of a lock mass standard.

An improved polymerase chain reaction-restriction fragment length polymorphism method for genotyping pufferfish species by liquid chromatography/mass spectrometry is described. A reverse-phase silica monolith column is employed for separating digested amplicons. This method can elucidate the monoisotopic masses of oligonucleotides, which is useful for identifying base composition.

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by ...

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3&#39; end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessment

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but especially on red blood cells (RBCs). As a receptor for the complement components C3b and C4b, CR1 regulates the activation of the complement cascade and promotes the phagocytosis of immune complexes and cellular debris, as well as the amyloid-beta (A&#946;) peptide in Alzheimer's disease (AD). Several studies have confirmed AD-associated single nucleotide polymorphisms (SNPs), as well as a copy-number variation (CNV) in the CR1 gene. Here, we describe an innovative method for determining the length polymorphism of the CR1 receptor. The receptor includes three domains, called long homologous repeats (LHR)-LHR-A, LHR-C, and LHR-D-and an n domain, LHR-B, where n is an integer between 0 and 3. Using a single pair of specific primers, the genetic material is used to amplify a first fragment of the LHR-B domain (the variant amplicon B) and a second fragment of the LHR-C domain (the invariant amplicon). The variant amplicon B and the invariant amplicon display differences at five nucleotides outside of the hybridization areas of said primers. The numbers of variant amplicons B and of invariant amplicons is deduced using a quantitative tool (high-resolution melting (HRM) curves), and the ratio of the variant amplicon B to the invariant amplicon differs according to the CR1 length polymorphism. This method provides several advantages over the canonical phenotype method, as it does not require fresh material and is cheaper, faster, and therefore applicable to larger populations. Thus, the use of this method should be helpful to better understand the role of CR1 isoforms in the pathogenesis of diseases such as AD.

Here, we describe an innovative method to determine complement receptor 1 (CR1) length polymorphisms for use in several applications, particularly the assessment of susceptibility to diseases such as Alzheimer&#39;s disease (AD). This method could be useful to better understand the role of CR1 isoforms in the pathogenesis of AD.

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The mechanisms of GA and ABA signaling can be interrogated by introducing reporter gene constructs into aleurone cells via particle bombardment, with the resulting transient expression measured using enzyme assays. An improved protocol is reported that partially automates and streamlines the grain homogenization step and the enzyme assays, allowing significantly more throughput than existing methods. Homogenization of the grain samples is carried out using an automated tissue homogenizer, and GUS (&#946;-glucuronidase) assays are carried out using a 96-well plate system. Representative results using the protocol suggest that phospholipase D activity may play an important role in the activation of HVA1 gene expression by ABA, through the transcription factor TaABF1.

An improved protocol is presented for the measurement of transient gene expression from reporter constructs in barley aleurone cells after particle bombardment. The combination of automated grain grinding with 96-well plate enzyme assays provides high throughput for the procedure.

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

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

Summary

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Chapters in this video

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics (MSPP)

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessment

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput