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	<title>PCR | Visikol</title>
	<atom:link href="https://visikol.com/blog/tag/pcr/feed/" rel="self" type="application/rss+xml" />
	<link>https://visikol.com</link>
	<description>Advanced Drug Discovery and Bioimaging Services</description>
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		<title>Gene-specific methylation analysis by LC-MS/MS</title>
		<link>https://visikol.com/blog/2022/08/03/gene-specific-methylation/</link>
		
		<dc:creator><![CDATA[Carol Tomaszewski]]></dc:creator>
		<pubDate>Wed, 03 Aug 2022 13:01:51 +0000</pubDate>
				<category><![CDATA[Blogs]]></category>
		<category><![CDATA[Featured]]></category>
		<category><![CDATA[Latest Blogs]]></category>
		<category><![CDATA[biomarker]]></category>
		<category><![CDATA[bisulfite conversion]]></category>
		<category><![CDATA[cell painting]]></category>
		<category><![CDATA[drug discovery]]></category>
		<category><![CDATA[epigenetic]]></category>
		<category><![CDATA[Gene specific methylation]]></category>
		<category><![CDATA[gene-methylation]]></category>
		<category><![CDATA[gene-promoter methylation]]></category>
		<category><![CDATA[Global DNA methylation]]></category>
		<category><![CDATA[LC-MS/MS]]></category>
		<category><![CDATA[P53]]></category>
		<category><![CDATA[PCR]]></category>
		<category><![CDATA[tumor suppressive genes]]></category>
		<guid isPermaLink="false">https://visikol.com/?p=18042</guid>

					<description><![CDATA[DNA methylation is a critical epigenetic modification that controls gene expression and genome stability, which has oncogenic implications during cancer initiation. DNA methylation occurs mainly on the CpG islands, which are mainly enriched at gene promoters. Hypermethylated promoters have been shown to silence the corresponding gene expression, while hypomethylation has the opposite effect. Interestingly,  [...]]]></description>
										<content:encoded><![CDATA[<div class="fusion-fullwidth fullwidth-box fusion-builder-row-1 fusion-flex-container nonhundred-percent-fullwidth non-hundred-percent-height-scrolling" style="--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;" ><div class="fusion-builder-row fusion-row fusion-flex-align-items-flex-start fusion-flex-content-wrap" style="max-width:1216.8px;margin-left: calc(-4% / 2 );margin-right: calc(-4% / 2 );"><div class="fusion-layout-column fusion_builder_column fusion-builder-column-0 fusion_builder_column_1_1 1_1 fusion-flex-column" style="--awb-bg-size:cover;--awb-width-large:100%;--awb-margin-top-large:0px;--awb-spacing-right-large:1.92%;--awb-margin-bottom-large:30px;--awb-spacing-left-large:1.92%;--awb-width-medium:100%;--awb-order-medium:0;--awb-spacing-right-medium:1.92%;--awb-spacing-left-medium:1.92%;--awb-width-small:100%;--awb-order-small:0;--awb-spacing-right-small:1.92%;--awb-spacing-left-small:1.92%;"><div class="fusion-column-wrapper fusion-column-has-shadow fusion-flex-justify-content-flex-start fusion-content-layout-column"><div class="fusion-text fusion-text-1"><p>DNA methylation is a critical epigenetic modification that controls gene expression and genome stability, which has oncogenic implications during cancer initiation. DNA methylation occurs mainly on the CpG islands, which are mainly enriched at gene promoters. Hypermethylated promoters have been shown to silence the corresponding gene expression, while hypomethylation has the opposite effect. Interestingly, cancer is associated with mixed DNA methylation dysregulation, which includes a genome-wide hypomethylation and hypermethylation associated with the silencing of vital tumor-suppressor genes.[i] Retrieving gene-methylation patterns is an important biomarker for early cancer prediction and other diseases.</p>
<p>Whole-genome bisulfite sequencing is a well-established protocol for detecting methylated cytosines in genomic DNA. This protocol uses bisulfite-based deamination of unmethylated cytosine into uracil bases, while the methylated cytosine bases are unaffected. During sequencing, uracil bases are deaminated into thymidine, while methylated cytosines, which resisted the deamination step, are sequenced into cytosines (Figure 1A). Site-specific quantification of the generated cytosines provides information on the methylation status of the selected DNA region. One of the tools that can be used to retrieve such information is the LC-MS/MS.</p>
</div><div class="fusion-image-element " style="text-align:center;--awb-caption-title-font-family:var(--h2_typography-font-family);--awb-caption-title-font-weight:var(--h2_typography-font-weight);--awb-caption-title-font-style:var(--h2_typography-font-style);--awb-caption-title-size:var(--h2_typography-font-size);--awb-caption-title-transform:var(--h2_typography-text-transform);--awb-caption-title-line-height:var(--h2_typography-line-height);--awb-caption-title-letter-spacing:var(--h2_typography-letter-spacing);"><span class=" fusion-imageframe imageframe-none imageframe-1 hover-type-none"><img fetchpriority="high" decoding="async" width="1236" height="616" alt="bisulfite conversion-PCR sequence" title="bisulfite conversion-PCR sequence" src="https://visikol.com/wp-content/uploads/2022/07/figure1.png" class="img-responsive wp-image-18070" srcset="https://visikol.com/wp-content/uploads/2022/07/figure1-200x100.png 200w, https://visikol.com/wp-content/uploads/2022/07/figure1-400x199.png 400w, https://visikol.com/wp-content/uploads/2022/07/figure1-600x299.png 600w, https://visikol.com/wp-content/uploads/2022/07/figure1-800x399.png 800w, https://visikol.com/wp-content/uploads/2022/07/figure1-1200x598.png 1200w, https://visikol.com/wp-content/uploads/2022/07/figure1.png 1236w" sizes="(max-width: 1024px) 100vw, (max-width: 640px) 100vw, 1200px" /></span></div><div class="fusion-text fusion-text-2 fusion-text-no-margin" style="--awb-font-size:12px;--awb-margin-bottom:15px;"><p><em><strong>Figure 1.</strong> <strong>A)</strong> Overview of the bisulfite conversion-PCR sequence. <strong>B)</strong> LC-MS/MS selected base peak chromatogram of dC (top) at m/z 228.1/112.1 and 5mdC at m/z 242.1/126.1 (bottom) obtained from the enzymatic hydrolysis of 150 ng of the P53-amplified amplicon via DNA Degradase Plus (Zymo Research, E2021). As expected, 5mdC is not detected, which was lost during the bisulfite conversion step. Instead, a strong peak of dC was present in the P53 amplicon which is equivalent to the original methylation status of that gene.</em></p>
</div><div class="fusion-text fusion-text-3"><p>Visikol has recently developed a sensitive bioanalytical method for the quantification of global DNA methylation by LC-MS/MS, which is based on the enzymatic hydrolysis of the genomic DNA and the simultaneous quantification of the corresponding nucleotides, 5-methyl-2’-deoxycytidine (5mdC), and 2’-deoxycytidine (dC), using the QTRAP 4000 mass spectrometer. <a href="https://visikol.com/blog/2022/03/29/quantification-of-global-dna-methylation-status-by-lc-ms-ms/">This method was used to measure the changes in the global DNA methylation status of two cancer cell lines</a>, A549 (human lung carcinoma) and HCT116 (human colorectal carcinoma), following the treatment with 5 µM of the DNA methyltransferase inhibitor 5-azacytidine (5-AZA). Using the DNA samples extracted from the 5-AZA-treated HCT116 cancer cell line, we extended the applicability of our method to quantify DNA methylation of the promoter region of the tumor suppressor gene, the p53 gene. Briefly, DNA samples were bisulfite converted, which then underwent a PCR amplification based on the following primers: ATTGTTTAGTTTTGTGTTAGGAGTTT, forward strand, and TCAATCAAAAACTTACCCAATCC for the reverse strand. The amplicons were then purified, subjected to our optimized enzymatic hydrolysis protocol, and finally analyzed by LC-MS/MS (Figure 1). The molar concentration of generated cytosine bases in the PCR amplicon is equivalent to what is expected based on the original methylation status of that gene. As expected, the amplicon hydrolysate showed no deoxythymidine, dT at sites where dC was anticipated. This data showed that the methylation status of the p53 gene in the A549 and HCT116 cancer cells did not change after the treatment with 5-AZA for 72 hours. Interestingly, 5-AZA treatment induced aggregation of nuclear material and decrease in the cell organelles (Figure 2).</p>
</div><div class="fusion-image-element " style="text-align:center;--awb-caption-title-font-family:var(--h2_typography-font-family);--awb-caption-title-font-weight:var(--h2_typography-font-weight);--awb-caption-title-font-style:var(--h2_typography-font-style);--awb-caption-title-size:var(--h2_typography-font-size);--awb-caption-title-transform:var(--h2_typography-text-transform);--awb-caption-title-line-height:var(--h2_typography-line-height);--awb-caption-title-letter-spacing:var(--h2_typography-letter-spacing);"><span class=" fusion-imageframe imageframe-none imageframe-2 hover-type-none"><img decoding="async" width="800" height="402" alt="Cell painting of HCT116 cancer cells" title="Cell painting of HCT116 cancer cells" src="https://visikol.com/wp-content/uploads/2022/07/figure2-800x402.png" class="img-responsive wp-image-18071" srcset="https://visikol.com/wp-content/uploads/2022/07/figure2-200x101.png 200w, https://visikol.com/wp-content/uploads/2022/07/figure2-400x201.png 400w, https://visikol.com/wp-content/uploads/2022/07/figure2-600x302.png 600w, https://visikol.com/wp-content/uploads/2022/07/figure2-800x402.png 800w, https://visikol.com/wp-content/uploads/2022/07/figure2.png 1199w" sizes="(max-width: 1024px) 100vw, (max-width: 640px) 100vw, 800px" /></span></div><div class="fusion-text fusion-text-4 fusion-text-no-margin" style="--awb-font-size:12px;--awb-margin-bottom:15px;"><p><em><strong>Figure 2. Cell painting of HCT116 cancer cells: </strong>Control and 5-AZA treated cells are  stained with Hoechst (blue), Concanavalin A (green), SYTO14 (Magenta), WGA (yellow), and Mito tracker (deep red).  5-Aza shows aggregation of nuclear material and inhibition of cellular components.</em></p>
</div><div class="fusion-text fusion-text-5"><h4><strong>If you are working on an epigenetic <a href="https://visikol.com/areas/">drug discovery project</a> and need help quantifying your global DNA methylation or a gene-specific methylation, <a href="https://visikol.com/get-started-today/">please reach out to our team to explore your epigenetic needs.</a></strong></h4>
</div><div class="fusion-separator fusion-full-width-sep" style="align-self: center;margin-left: auto;margin-right: auto;width:100%;"><div class="fusion-separator-border sep-single sep-solid" style="--awb-height:20px;--awb-amount:20px;border-color:#e0dede;border-top-width:1px;"></div></div><div class="fusion-text fusion-text-6" style="--awb-font-size:12px;--awb-margin-top:15px;"><p><strong>Reference</strong>:   <a href="#_ednref1">[i]</a> Skvortsova, K.; Stirzaker, C.; Taberlay, P. The DNA methylation landscape in cancer. <em>Essays Biochem.</em>, <strong>2019</strong>, <em>6</em>, 797-811.</p>
</div></div></div></div></div>The post <a href="https://visikol.com/blog/2022/08/03/gene-specific-methylation/">Gene-specific methylation analysis by LC-MS/MS</a> first appeared on <a href="https://visikol.com">Visikol</a>.]]></content:encoded>
					
		
		
			</item>
		<item>
		<title>Examining gene expression with RT-qPCR</title>
		<link>https://visikol.com/blog/2021/05/02/examining-gene-expression-with-rt-qpcr/</link>
					<comments>https://visikol.com/blog/2021/05/02/examining-gene-expression-with-rt-qpcr/#comments</comments>
		
		<dc:creator><![CDATA[Griffin Ferrara]]></dc:creator>
		<pubDate>Sun, 02 May 2021 14:18:53 +0000</pubDate>
				<category><![CDATA[Blogs]]></category>
		<category><![CDATA[Featured]]></category>
		<category><![CDATA[gene expression]]></category>
		<category><![CDATA[gene expression assay]]></category>
		<category><![CDATA[PCR]]></category>
		<category><![CDATA[pcr assay]]></category>
		<category><![CDATA[qPCR]]></category>
		<category><![CDATA[qPCR assay]]></category>
		<category><![CDATA[RT-qPCR]]></category>
		<guid isPermaLink="false">https://visikol.com/?p=14796</guid>

					<description><![CDATA[At Visikol, our services have grown from our expertise in cell culture, high-content tissue imaging, and advanced digital pathology.  For nearly a year, however, our in vitro services have been augmented by gene expression analyses that allow us to provide clients with a more complete overview of the often complex interactions lurking behind the  [...]]]></description>
										<content:encoded><![CDATA[<p><div class="fusion-fullwidth fullwidth-box fusion-builder-row-2 fusion-flex-container nonhundred-percent-fullwidth non-hundred-percent-height-scrolling" style="--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;" ><div class="fusion-builder-row fusion-row fusion-flex-align-items-flex-start fusion-flex-content-wrap" style="max-width:1216.8px;margin-left: calc(-4% / 2 );margin-right: calc(-4% / 2 );"><div class="fusion-layout-column fusion_builder_column fusion-builder-column-1 fusion_builder_column_1_1 1_1 fusion-flex-column" style="--awb-bg-size:cover;--awb-width-large:100%;--awb-margin-top-large:0px;--awb-spacing-right-large:1.92%;--awb-margin-bottom-large:30px;--awb-spacing-left-large:1.92%;--awb-width-medium:100%;--awb-order-medium:0;--awb-spacing-right-medium:1.92%;--awb-spacing-left-medium:1.92%;--awb-width-small:100%;--awb-order-small:0;--awb-spacing-right-small:1.92%;--awb-spacing-left-small:1.92%;"><div class="fusion-column-wrapper fusion-column-has-shadow fusion-flex-justify-content-flex-start fusion-content-layout-column"><div class="fusion-text fusion-text-7"><p>At Visikol, our services have grown from our expertise in cell culture, high-content tissue imaging, and advanced digital pathology.  For nearly a year, however, our <em>in vitro </em>services have been augmented by gene expression analyses that allow us to provide clients with a more complete overview of the often complex interactions lurking behind the results produced by drug candidates.  These assays center around quantitative (real-time) reverse transcription polymerase chain reaction, thankfully known day to day as RT-qPCR.</p>
<p>The polymerase chain reaction’s common nickname of “PCR” has worked its way into conversation for many around the world during the course of the global coronavirus pandemic.  PCR’s use in COVID-19 testing has granted it a recognizable place in public medical awareness alongside mainstay three-letter abbreviations like MRI, ACL, and UTI.  However, while it may come up at the dinner table a lot more often these days, PCR in various forms has been at the core of molecular biology for decades and truly revolutionized the field after its invention by Kary Mullis in 1983.  (Mullis would go on to win the Nobel Prize in Chemistry a decade later, and the story of his PCR “revelation” has become a part of modern scientific folklore.  Seriously, look it up sometime…!)</p>
<p>The concepts that form the foundation of PCR are deceptively simple.  With minimal resources – basically a few simple reagents and a way to control their temperature – researchers are able to take any sequence of DNA and recreate it billions of times, essentially generating as many copies of a sequence of interest as they desire.  The process is fast, consistent, relatively inexpensive…and undeniably beautiful.  Indeed, Dr. Mullis was like Lennon and McCartney in a lab coat, reaching into thin air and pulling out something magical that had always existed but was just waiting to be put in order.</p>
<p>Variations on the core process of PCR have led to a long list of modern applications for the platform.  At Visikol, we make extensive use of RT-qPCR to provide clients with a full picture of the effects of their test compounds.  This expansion of PCR’s capabilities adds the use of a reverse transcriptase (the “RT” in the title) to generate a DNA template from RNA samples, and its quantitative/real-time aspect (the “q”) involves step-by-step monitoring of PCR products to determine of the amount of target DNA that began the process.  Coupled with the capture of messenger RNA at critical points during the action of clients’ compounds of interest, RT-qPCR gives us a clearer picture of a drug’s impact by showing us the changes in gene expression elicited by its addition to cells.</p>
<p>If your company is looking for a way to solidify your understanding of a drug’s effects by complementing traditional histological and imaging techniques – or even for a first-line assessment of a compound’s impact on your tissue of interest – RT-qPCR may be the technique you’re looking for.  Our scientists can work with your requirements and budget to determine what experimental design and RT-qPCR protocol will work best for your needs.  Whether it’s for cancer drug efficacy, liver disease pathology, or a model with less well-defined gene expression parameters, we can help elaborate on the molecular phenomena lying beneath the complex mechanisms of your drug candidates.</p>
</div></div></div></div></div><div class="fusion-fullwidth fullwidth-box fusion-builder-row-3 fusion-flex-container nonhundred-percent-fullwidth non-hundred-percent-height-scrolling" style="--awb-border-radius-top-left:0px;--awb-border-radius-top-right:0px;--awb-border-radius-bottom-right:0px;--awb-border-radius-bottom-left:0px;--awb-flex-wrap:wrap;" ><div class="fusion-builder-row fusion-row fusion-flex-align-items-flex-start fusion-flex-content-wrap" style="max-width:1216.8px;margin-left: calc(-4% / 2 );margin-right: calc(-4% / 2 );"><div class="fusion-layout-column fusion_builder_column fusion-builder-column-2 fusion_builder_column_1_1 1_1 fusion-flex-column" style="--awb-bg-size:cover;--awb-width-large:100%;--awb-margin-top-large:0px;--awb-spacing-right-large:1.92%;--awb-margin-bottom-large:30px;--awb-spacing-left-large:1.92%;--awb-width-medium:100%;--awb-order-medium:0;--awb-spacing-right-medium:1.92%;--awb-spacing-left-medium:1.92%;--awb-width-small:100%;--awb-order-small:0;--awb-spacing-right-small:1.92%;--awb-spacing-left-small:1.92%;"><div class="fusion-column-wrapper fusion-column-has-shadow fusion-flex-justify-content-flex-start fusion-content-layout-column"><div class="fusion-image-element " style="--awb-caption-title-font-family:var(--h2_typography-font-family);--awb-caption-title-font-weight:var(--h2_typography-font-weight);--awb-caption-title-font-style:var(--h2_typography-font-style);--awb-caption-title-size:var(--h2_typography-font-size);--awb-caption-title-transform:var(--h2_typography-text-transform);--awb-caption-title-line-height:var(--h2_typography-line-height);--awb-caption-title-letter-spacing:var(--h2_typography-letter-spacing);"><span class=" fusion-imageframe imageframe-none imageframe-3 hover-type-none"><img decoding="async" width="1024" height="512" title="ampplotpic" src="https://visikol.com/wp-content/uploads/2021/04/ampplotpic-1024x512.png" alt class="img-responsive wp-image-14804" srcset="https://visikol.com/wp-content/uploads/2021/04/ampplotpic-200x100.png 200w, https://visikol.com/wp-content/uploads/2021/04/ampplotpic-400x200.png 400w, https://visikol.com/wp-content/uploads/2021/04/ampplotpic-600x300.png 600w, https://visikol.com/wp-content/uploads/2021/04/ampplotpic-800x400.png 800w, https://visikol.com/wp-content/uploads/2021/04/ampplotpic.png 1200w" sizes="(max-width: 1024px) 100vw, (max-width: 640px) 100vw, 1024px" /></span></div></div></div></div></div></p>The post <a href="https://visikol.com/blog/2021/05/02/examining-gene-expression-with-rt-qpcr/">Examining gene expression with RT-qPCR</a> first appeared on <a href="https://visikol.com">Visikol</a>.]]></content:encoded>
					
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