{"id":9272,"date":"2015-08-19T20:05:44","date_gmt":"2015-08-19T20:05:44","guid":{"rendered":"http:\/\/admin.acceleratingscience.com\/behindthebench\/?p=9272"},"modified":"2020-05-28T19:20:35","modified_gmt":"2020-05-28T19:20:35","slug":"copy-number-variation-how-does-it-work-asktaqman-34","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/copy-number-variation-how-does-it-work-asktaqman-34\/","title":{"rendered":"Copy Number Variation &#8211; How does it work? AskTaqMan #34"},"content":{"rendered":"<p>Genotyping single nucleotide polymorphisms, or SNPs, using TaqMan Assays has been very well established.\u00a0 But have you heard about copy number variations?<\/p>\n<p><a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/pcr\/real-time-pcr\/real-time-pcr-assays\/cnv-analysis-using-taqman.html?icid=lsg_gsd_CP1188_blog_asktaqman\" target=\"_blank\" rel=\"noopener noreferrer\">Copy number variation (CNV)<\/a> is a type of structural variation that occurs when a DNA segment of 1 kb to several megabases in length is present in variable copy numbers compared to a reference genome. These CNVs can influence gene expression and can be associated with specific phenotypes and diseases. This variation covers approximately 12% of the human genome and includes deletions and duplications. <a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/pcr\/real-time-pcr\/real-time-pcr-assays\/cnv-analysis-using-taqman.html?icid=lsg_gsd_CP1188_blog_asktaqman\" target=\"_blank\" rel=\"noopener noreferrer\">TaqMan Copy Number assays<\/a> offer a targeted approach combining high accuracy, specificity, ease of use and sample throughput, to validate copy number changes or screen large sample sets.<\/p>\n<p><iframe loading=\"lazy\" width=\"760\" height=\"428\" src=\"https:\/\/www.youtube.com\/embed\/QSenRqbEABM?feature=oembed\" frameborder=\"0\" allow=\"accelerometer; autoplay; encrypted-media; gyroscope; picture-in-picture\" allowfullscreen><\/iframe><\/p>\n<p class=\"TableParagraph\">So how does this all work?<\/p>\n<p class=\"TableParagraph\"><strong>Let\u2019s take a look at our lab book<\/strong><\/p>\n<p>TaqMan Copy Number assays are a duplex experiment using a FAM labeled target assay and a VIC labeled reference assay in the same well to quantify small fold changes. The reference assay targets a gene of known and stable copy number. For example, RNase P is often used for human samples, as it has been shown to be present and stable at 2 copies.<\/p>\n<p>To determine copy number, a relative quantitation analysis is performed between the unknown sample and a calibrator sample (one in which the copy number of the target gene is known).<\/p>\n<p>The sample is combined with both assays, master mix, and then run in quadruplicate in the real-time PCR instrument.<\/p>\n<p>Let\u2019s look at an example in which the target gene is on the X-chromosome. In a male sample, we should then find a copy number of 1 for the target.<\/p>\n<p class=\"TableParagraph\">A baseline subtracted cycle threshold or dCt is determined between the two assays. In our example, the unknown sample has a dCt of 1.5 And the calibrator a dCt of 0.5.<\/p>\n<p class=\"TableParagraph\">We then subtract the dCt of the calibrator from the dCt of the unknown.<\/p>\n<p class=\"TableParagraph\">Next we convert this value to a relative quantity<\/p>\n<p class=\"TableParagraph\">And then multiply the relative quantity or RQ by the copy number of the calibrator, which in this case are 2 with RnaseP.<\/p>\n<p>Ah-ha! A copy of 1. Wasn\u2019t that easy?\u00a0 And no messy standard curves to deal with.<\/p>\n<p>Thermo Fisher Scientific offers a free software program which will do all of the analysis for you with the exported data from a compatible instrument. The software provides statistics to help evaluate data confidence and can calculate copy number even when a calibrator sample is not used.<\/p>\n<p>So there you have it!\u00a0 A complete workflow solution for measuring DNA copy number variation in a precise quantitative fashion. Whether you need to quantify copy numbers of important pharmacogenomics genes such as CYP2D6, or you\u2019re investigating copy number variants underlying cancer, Applied Biosystems has the reagents and tools to drive your research.<\/p>\n<p>If you\u2019ve got more qPCR or digital PCR questions, remember, just ask Taqman.\u00a0 Submit your question at <a href=\"http:\/\/www.thermofisher.com\/us\/en\/home\/products-and-services\/promotions\/ask.html?cid=social_btb_genequant\" target=\"_blank\" rel=\"nofollow noopener noreferrer\">thermofisher.com\/ask<\/a><\/p>\n","protected":false},"excerpt":{"rendered":"<p>Genotyping single nucleotide polymorphisms, or SNPs, using TaqMan Assays has been very well established.\u00a0 But have you heard about copy number variations? Copy number variation (CNV) is a type of structural variation that occurs when a DNA segment of 1 kb to several megabases in length is present in variable copy numbers compared to a<\/p>\n","protected":false},"author":112,"featured_media":9273,"comment_status":"open","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_kad_blocks_custom_css":"","_kad_blocks_head_custom_js":"","_kad_blocks_body_custom_js":"","_kad_blocks_footer_custom_js":"","_monsterinsights_skip_tracking":false,"_genesis_hide_title":false,"_genesis_hide_breadcrumbs":false,"_genesis_hide_singular_image":false,"_genesis_hide_footer_widgets":false,"_genesis_custom_body_class":"","_genesis_custom_post_class":"","_genesis_layout":"","_jetpack_newsletter_access":"","_jetpack_dont_email_post_to_subs":false,"_jetpack_newsletter_tier_id":0,"_jetpack_memberships_contains_paywalled_content":false,"_jetpack_memberships_contains_paid_content":false,"footnotes":""},"categories":[108],"tags":[17,18,153,43],"division":[],"class_list":{"0":"post-9272","1":"post","2":"type-post","3":"status-publish","4":"format-standard","5":"has-post-thumbnail","7":"category-lab-tips-and-tricks","8":"tag-applied-biosystems","9":"tag-ask-taqman","10":"tag-genomic-variants","11":"tag-pcr","12":"entry"},"_selected_authors":"","_selected_reviewers":"","acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO Premium plugin v27.8 (Yoast SEO v27.8) - https:\/\/yoast.com\/product\/yoast-seo-premium-wordpress\/ -->\n<title>Copy Number Variation - How does it work? 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Copy number variation (CNV) is a type of structural variation that occurs when a DNA segment of 1 kb to several megabases in length is present in variable copy numbers compared to a\" \/>\n<meta property=\"og:url\" content=\"https:\/\/www.thermofisher.com\/blog\/behindthebench\/copy-number-variation-how-does-it-work-asktaqman-34\/\" \/>\n<meta property=\"og:site_name\" content=\"Behind the Bench\" \/>\n<meta property=\"article:publisher\" content=\"https:\/\/www.facebook.com\/thermofisher\" \/>\n<meta property=\"article:published_time\" content=\"2015-08-19T20:05:44+00:00\" \/>\n<meta property=\"article:modified_time\" content=\"2020-05-28T19:20:35+00:00\" \/>\n<meta property=\"og:image\" content=\"https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2016\/11\/ask.jpg\" \/>\n\t<meta property=\"og:image:width\" content=\"1570\" \/>\n\t<meta property=\"og:image:height\" content=\"876\" \/>\n\t<meta property=\"og:image:type\" content=\"image\/jpeg\" \/>\n<meta name=\"author\" content=\"Mike Troutman\" \/>\n<meta name=\"twitter:card\" content=\"summary_large_image\" \/>\n<meta name=\"twitter:creator\" content=\"@thermofisher\" \/>\n<meta name=\"twitter:site\" content=\"@thermofisher\" \/>\n<meta name=\"twitter:label1\" content=\"Written by\" \/>\n\t<meta name=\"twitter:data1\" content=\"Mike Troutman\" \/>\n\t<meta name=\"twitter:label2\" content=\"Est. reading time\" \/>\n\t<meta name=\"twitter:data2\" content=\"2 minutes\" \/>\n<script type=\"application\/ld+json\" class=\"yoast-schema-graph\">{\"@context\":\"https:\\\/\\\/schema.org\",\"@graph\":[{\"@type\":\"Article\",\"@id\":\"https:\\\/\\\/www.thermofisher.com\\\/blog\\\/behindthebench\\\/copy-number-variation-how-does-it-work-asktaqman-34\\\/#article\",\"isPartOf\":{\"@id\":\"https:\\\/\\\/www.thermofisher.com\\\/blog\\\/behindthebench\\\/copy-number-variation-how-does-it-work-asktaqman-34\\\/\"},\"author\":{\"name\":\"Mike Troutman\",\"@id\":\"https:\\\/\\\/admin.acceleratingscience.com\\\/behindthebench\\\/#\\\/schema\\\/person\\\/4d15e6a9e79225dd37a71a333d816e64\"},\"headline\":\"Copy Number Variation &#8211; How does it work? 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