How to Analyze Gene Expression with RNA-seq Data

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Odoi","image":"https://media.licdn.com/dms/image/v2/D4E03AQHGqanhqOtp1g/profile-displayphoto-shrink_400_400/profile-displayphoto-shrink_400_400/0/1707468511927?e=2147483647&v=beta&t=Gr8dzqgkMgPs8usgA7s45JtYkdRTU5SBGH88xk8_zBI","interactionStatistic":{"@type":"InteractionCounter","interactionType":"https://schema.org/FollowAction","name":"Followers","userInteractionCount":0}},{"@type":"Person","url":"https://www.linkedin.com/in/iftekhar-rafiqullah-phd-8064a962","name":"Iftekhar Rafiqullah, PhD","interactionStatistic":{"@type":"InteractionCounter","interactionType":"https://schema.org/FollowAction","name":"Followers","userInteractionCount":0}},{"@type":"Person","url":"https://www.linkedin.com/in/teawhale","name":"Jingbo Zhang","image":"https://media.licdn.com/dms/image/v2/C5603AQHak-ZrTQfMCw/profile-displayphoto-shrink_400_400/profile-displayphoto-shrink_400_400/0/1657307265107?e=2147483647&v=beta&t=kDwj4XWqzuGeNRJPFNILYUovxezWZE0ogNx4onDL6-0","interactionStatistic":{"@type":"InteractionCounter","interactionType":"https://schema.org/FollowAction","name":"Followers","userInteractionCount":0}},{"@type":"Person","url":"https://uk.linkedin.com/in/frances-turner-7797bb233","name":"Frances Turner","image":"https://media.licdn.com/dms/image/v2/D4E03AQFq2Pq4AVakoQ/profile-displayphoto-shrink_400_400/profile-displayphoto-shrink_400_400/0/1721740335702?e=2147483647&v=beta&t=HP42P8LJDv120cm55tJQKjTJE6oRq1yKVKpN7R69a50","interactionStatistic":{"@type":"InteractionCounter","interactionType":"https://schema.org/FollowAction","name":"Followers","userInteractionCount":0}},{"@type":"Person","url":"https://www.linkedin.com/in/salma-ben-salem15","name":"Salma B.","image":"https://media.licdn.com/dms/image/v2/D5603AQGUo0XPa8-3uA/profile-displayphoto-shrink_400_400/profile-displayphoto-shrink_400_400/0/1689386484141?e=2147483647&v=beta&t=OENoLMAdipLfanT12zG1KSjOIeVb2jmkpU4dl_qn3pk","interactionStatistic":{"@type":"InteractionCounter","interactionType":"https://schema.org/FollowAction","name":"Followers","userInteractionCount":0}}],"comment":[{"@type":"Comment","text":"The preparation of samples and sequencing libraries is a critical step in utilizing RNA-seq data for gene expression analysis. \nThis process involves converting RNA molecules into sequencer-ready libraries, ensuring accurate representation of gene expression levels. \nProper sample preparation enhances the reliability and reproducibility of RNA-seq results, allowing researchers to draw meaningful insights into gene expression patterns. \nThis meticulous step is foundational for unlocking the full potential of RNA-seq data and gaining a comprehensive understanding of the dynamic landscape of gene expression in biological systems.","upVoteCount":2,"author":{"@type":"Person","url":"https://gh.linkedin.com/in/dennisodoi","name":"Dennis Odoi","image":"https://media.licdn.com/dms/image/v2/D4E03AQHGqanhqOtp1g/profile-displayphoto-shrink_400_400/profile-displayphoto-shrink_400_400/0/1707468511927?e=2147483647&v=beta&t=Gr8dzqgkMgPs8usgA7s45JtYkdRTU5SBGH88xk8_zBI","interactionStatistic":{"@type":"InteractionCounter","interactionType":"https://schema.org/FollowAction","name":"Followers","userInteractionCount":0}}},{"@type":"Comment","text":"I agree. Sample preparation is the first critical step. Extraction of RNA from biological samples needs to be done carefully under the hood so that it doesn’t get contaminated. Kits and reagents need to be separated. Once cDNA is ready, keep continuing the protocol for fragmentation, adapter ligation, etc. Make sure QC is done to check concentrations, size , etc. appropriate for sequencing breadth and depth.","upVoteCount":0,"author":{"@type":"Person","url":"https://www.linkedin.com/in/iftekhar-rafiqullah-phd-8064a962","name":"Iftekhar Rafiqullah, PhD","interactionStatistic":{"@type":"InteractionCounter","interactionType":"https://schema.org/FollowAction","name":"Followers","userInteractionCount":0}}},{"@type":"Comment","text":"If the whole genome date is not available for the organism you studied, you can also do the De novo transcriptome assembly from the RNA-seq data you received. The reads can then align to the De novo transcriptome.","upVoteCount":3,"author":{"@type":"Person","url":"https://www.linkedin.com/in/teawhale","name":"Jingbo Zhang","image":"https://media.licdn.com/dms/image/v2/C5603AQHak-ZrTQfMCw/profile-displayphoto-shrink_400_400/profile-displayphoto-shrink_400_400/0/1657307265107?e=2147483647&v=beta&t=kDwj4XWqzuGeNRJPFNILYUovxezWZE0ogNx4onDL6-0","interactionStatistic":{"@type":"InteractionCounter","interactionType":"https://schema.org/FollowAction","name":"Followers","userInteractionCount":0}}},{"@type":"Comment","text":"Sometimes more than one species is present in a sample (for instance a host and a pathogen). Even if you are only interested in one species, it is best practice to concatenate the the genomes/transcriptomes of all the species in your samples, then align all data to the concatenated genome. \nOnce you have performed the alignments, further QC can be carried out. We find the % of reads mapping to the genome/transcriptome, median PCR duplicate rate per gene, and % of rRNA are all useful metrics to assess library quality and ensure appropriate downstream analysis. Picard tools is useful at this point.","upVoteCount":0,"author":{"@type":"Person","url":"https://uk.linkedin.com/in/frances-turner-7797bb233","name":"Frances Turner","image":"https://media.licdn.com/dms/image/v2/D4E03AQFq2Pq4AVakoQ/profile-displayphoto-shrink_400_400/profile-displayphoto-shrink_400_400/0/1721740335702?e=2147483647&v=beta&t=HP42P8LJDv120cm55tJQKjTJE6oRq1yKVKpN7R69a50","interactionStatistic":{"@type":"InteractionCounter","interactionType":"https://schema.org/FollowAction","name":"Followers","userInteractionCount":0}}},{"@type":"Comment","text":"Before testing for differential expression, it is important to remove lowly expressed genes. Some tools (such as DESeq2) will do this for you, other tools (such as edgeR) required you to do this yourself. Failure to perform this step will result in much less significant adjusted p-values (or false discovery rates) after multiple correction testing.\nIt is also important to consider any factors in your experiment (such a sample batch) that may influence gene expression, and ensure you control for these by including them in your design matrix","upVoteCount":2,"author":{"@type":"Person","url":"https://uk.linkedin.com/in/frances-turner-7797bb233","name":"Frances Turner","image":"https://media.licdn.com/dms/image/v2/D4E03AQFq2Pq4AVakoQ/profile-displayphoto-shrink_400_400/profile-displayphoto-shrink_400_400/0/1721740335702?e=2147483647&v=beta&t=HP42P8LJDv120cm55tJQKjTJE6oRq1yKVKpN7R69a50","interactionStatistic":{"@type":"InteractionCounter","interactionType":"https://schema.org/FollowAction","name":"Followers","userInteractionCount":0}}},{"@type":"Comment","text":"Analyzing RNA-seq data is a common method for estimating gene and transcript expression. The process typically involves several steps: Trimming, aligning, counting, and normalizing the sequenced reads, followed by differential expression (DE) analysis across different conditions. Statistical analysis is then performed to determine p-values and establish the false discovery rate (FDR) cutoff. Subsequent analyses depend on the specific hypothesis being investigated. Additional approaches may include utilizing Profiler, Gene Set Enrichment Analysis (GSEA), Cytoscape, EnrichmentMap, and Metascape for pathway enrichment analysis. These methods help provide mechanistic insights into the gene lists generated from RNA-Seq data.","upVoteCount":2,"author":{"@type":"Person","url":"https://www.linkedin.com/in/salma-ben-salem15","name":"Salma B.","image":"https://media.licdn.com/dms/image/v2/D5603AQGUo0XPa8-3uA/profile-displayphoto-shrink_400_400/profile-displayphoto-shrink_400_400/0/1689386484141?e=2147483647&v=beta&t=OENoLMAdipLfanT12zG1KSjOIeVb2jmkpU4dl_qn3pk","interactionStatistic":{"@type":"InteractionCounter","interactionType":"https://schema.org/FollowAction","name":"Followers","userInteractionCount":0}}}]} </script>  </head> <body dir="ltr">  <div class="guest-upsells"> <form class="google-auth base-google-auth" action="https://www.linkedin.com/uas/login-submit" method="post"> <input name="loginCsrfParam" value="c645173d-6972-44bb-8ec6-f9c48be6b255" type="hidden"> <input name="session_redirect" value="https://www.linkedin.com/advice/3/how-can-you-use-rna-seq-data-analyze-gene-rokle" type="hidden"> <input 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class="bg-color-surface"></div> </div> <div class="max-w-[1128px] py-2 md:py-4 mx-auto">  <code id="isGuest" style="display: none"></code> <code id="memberUrn" style="display: none"></code> <code id="isNbaRecommendedArticlesDeeplinkEnabled" style="display: none"></code> <code id="isDynamicTextareaEnabled" style="display: none"></code> <article class="article-main relative flex-grow">  <section class="core-section-container my-3 relative">    <div class="core-section-container__content break-words">  <div class="md:row pb-6 md:flex article-content-row-reduced"> <div class="column-article-segment-content md:column md:mb-8 md:mr-4 md:w-1/2 md:flex-1 md:pr-[88px]"> <div> <div class="article-segment-content-container ">  <section class="mb-2"> <ol itemscope itemtype="http://schema.org/BreadcrumbList" class="breadcrumbs "> <li itemprop="itemListElement" itemscope itemtype="http://schema.org/ListItem" class="breadcrumb inline-block mr-0.5 font-sans text-sm babybear:text-xs text-color-text after:content-['/'] after:text-color-text-low-emphasis after:ml-0.5 after:no-underline last:mr-0 last:after:hidden"> <a itemprop="item" class="breadcrumb__link inline-block link-no-visited-state !text-color-text-low-emphasis" href="https://www.linkedin.com/pulse/topics/home/" data-tracking-control-name="breadcrumb" data-tracking-will-navigate> <span class="font-normal" itemprop="name">All</span> </a> <meta itemprop="position" content="1"> </li> <li itemprop="itemListElement" itemscope itemtype="http://schema.org/ListItem" class="breadcrumb inline-block mr-0.5 font-sans text-sm babybear:text-xs text-color-text after:content-['/'] after:text-color-text-low-emphasis after:ml-0.5 after:no-underline last:mr-0 last:after:hidden"> <a aria-current="page" itemprop="item" class="breadcrumb__link inline-block link-no-visited-state !text-color-text" href="https://www.linkedin.com/pulse/topics/laboratory-techniques-s12858/" data-tracking-control-name="breadcrumb" data-tracking-will-navigate> <span class="font-semibold" itemprop="name">Laboratory Techniques</span> </a> <meta itemprop="position" content="2"> </li> </ol> </section> <h1 class="pulse-title text-display-md text-color-text lg:text-display-lg font-bold w-11/12 ">How can you use RNA-seq data to analyze gene expression?</h1>  <div class="powered-by-ai py-3 text-sm text-color-text-secondary">Powered by AI and the LinkedIn community</div> <section class="table-of-content pb-6"> <div class="group menu-item relative flex cursor-pointer flex-row justify-start border-0 pl-0" data-chunk-header-id="step-1:-prepare-your-samples-and-sequencing-libraries"> <h3 class="text-color-text group-hover:text-color-text-brand">1</h3> <div class="ml-2 mr-6 mt-0.5 min-w-[75%]"> <a class="fragmented-links font-normal !text-color-text underline decoration-1 underline-offset-4 group-hover:!text-color-text-brand" href="#step-1:-prepare-your-samples-and-sequencing-libraries" data-tracking-will-navigate 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data-tracking-control-name="article-ssr-frontend-x-article__toc-article-segment-navigation">Step 2: Perform sequencing and quality control </a> <p class="inline-promotion-text mt-0.5 text-xs font-normal text-color-text-low-emphasis">Be the first to add your personal experience</p> </div> <icon class="invisible absolute right-2 top-1/2 flex -translate-y-2/4 items-center justify-between rounded-full border-1 border-solid border-color-container-brand p-1 group-hover:visible" data-delayed-url="https://static.licdn.com/aero-v1/sc/h/6q8fxh9snmcwlh1wxg6fktwgf" data-svg-class-name="w-2 h-2 text-color-text-brand"></icon> </div> <div class="group menu-item relative flex cursor-pointer flex-row justify-start border-0 pl-0" data-chunk-header-id="step-3:-align-reads-to-a-reference-genome-or-transcriptome"> <h3 class="text-color-text group-hover:text-color-text-brand">3</h3> <div class="ml-2 mr-6 mt-0.5 min-w-[75%]"> <a class="fragmented-links font-normal !text-color-text underline decoration-1 underline-offset-4 group-hover:!text-color-text-brand" href="#step-3:-align-reads-to-a-reference-genome-or-transcriptome" data-tracking-will-navigate data-tracking-control-name="article-ssr-frontend-x-article__toc-article-segment-navigation">Step 3: Align reads to a reference genome or transcriptome </a>  </div> <icon class="invisible absolute right-2 top-1/2 flex -translate-y-2/4 items-center justify-between rounded-full border-1 border-solid border-color-container-brand p-1 group-hover:visible" data-delayed-url="https://static.licdn.com/aero-v1/sc/h/6q8fxh9snmcwlh1wxg6fktwgf" data-svg-class-name="w-2 h-2 text-color-text-brand"></icon> </div> <div class="group menu-item relative flex cursor-pointer flex-row justify-start border-0 pl-0" data-chunk-header-id="step-4:-perform-differential-expression-analysis"> <h3 class="text-color-text group-hover:text-color-text-brand">4</h3> <div class="ml-2 mr-6 mt-0.5 min-w-[75%]"> <a class="fragmented-links font-normal !text-color-text underline 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class="ml-2 mr-6 mt-0.5 min-w-[75%]"> <a class="fragmented-links font-normal !text-color-text underline decoration-1 underline-offset-4 group-hover:!text-color-text-brand" href="#step-6:-validate-and-extend-the-results" data-tracking-will-navigate data-tracking-control-name="article-ssr-frontend-x-article__toc-article-segment-navigation">Step 6: Validate and extend the results </a> <p class="inline-promotion-text mt-0.5 text-xs font-normal text-color-text-low-emphasis">Be the first to add your personal experience</p> </div> <icon class="invisible absolute right-2 top-1/2 flex -translate-y-2/4 items-center justify-between rounded-full border-1 border-solid border-color-container-brand p-1 group-hover:visible" data-delayed-url="https://static.licdn.com/aero-v1/sc/h/6q8fxh9snmcwlh1wxg6fktwgf" data-svg-class-name="w-2 h-2 text-color-text-brand"></icon> </div> <div class="group menu-item relative flex cursor-pointer flex-row justify-start border-0 pl-0" data-chunk-header-id="here’s-what-else-to-consider"> <h3 class="text-color-text group-hover:text-color-text-brand">7</h3> <div class="ml-2 mr-6 mt-0.5 min-w-[75%]"> <a class="fragmented-links font-normal !text-color-text underline decoration-1 underline-offset-4 group-hover:!text-color-text-brand" href="#here’s-what-else-to-consider" data-tracking-will-navigate data-tracking-control-name="article-ssr-frontend-x-article__toc-article-segment-navigation">Here’s what else to consider </a>  </div> <icon class="invisible absolute right-2 top-1/2 flex -translate-y-2/4 items-center justify-between rounded-full border-1 border-solid border-color-container-brand p-1 group-hover:visible" data-delayed-url="https://static.licdn.com/aero-v1/sc/h/6q8fxh9snmcwlh1wxg6fktwgf" data-svg-class-name="w-2 h-2 text-color-text-brand"></icon> </div> </section> <div class="x-article-introduction"> <div class="article-main__content" data-test-id="publishing-text-block"> <p> <span class>RNA-seq is a powerful technique to measure gene expression levels across different samples, conditions, or time points. It can reveal how genes are regulated, how they interact, and how they contribute to biological functions and phenotypes. However, RNA-seq data also pose many challenges, such as quality control, normalization, alignment, quantification, differential expression analysis, and interpretation. In this article, you will learn how to use RNA-seq data to analyze gene expression in six steps.</span> </p> </div> </div> </div> </div> </div> <div class="column-article-segment-contributions md:column md:w-1/2"> <code id="shouldExpandContributionBox" style="display: none"></code> <div class="x-featured-expert-section__wrapper flex h-full flex-1 flex-col"> <div class="x-featured-expert-section h-full"> <div class="x-featured-expert-section__article-experts"> <div class="x-featured-expert-section__title mt-2 pb-0.5 text-xl font-bold text-color-text"> Top experts in this article </div> <div class="x-featured-expert-section__subtitle pb-3 text-sm text-color-text-secondary"> Selected by the community from 6 contributions. <a href="https://www.linkedin.com/help/linkedin/answer/a1652832" target="_blank" aria-label="Learn more about collaborative articles" data-tracking-control-name="article-ssr-frontend-x-article__featured-expert-learn-more-link" data-tracking-will-navigate="true" 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id="content-urn_li_articleSegment__urn_li_linkedInArticle_7161790903102214144_7161790907422339072_"> <div class="article-main__content" data-test-id="publishing-text-block"> <h2 class="flex"> <a id="step-1:-prepare-your-samples-and-sequencing-libraries" href="#step-1:-prepare-your-samples-and-sequencing-libraries" class="dummy-anchor invisible absolute mt-[-100px]"></a> <span class="header-index pr-1.5">1</span> <span class>Step 1: Prepare your samples and sequencing libraries</span> </h2> </div> <div class="article-main__content" data-test-id="publishing-text-block"> <p> <span class>Before you can use RNA-seq data to analyze gene expression, you need to prepare your samples and sequencing libraries. This involves extracting RNA from your biological samples, such as cells, tissues, or organisms, and converting it into cDNA. Then, you need to fragment, size-select, and ligate adapters to the cDNA to generate sequencing libraries. The quality and quantity of your RNA and libraries will affect the accuracy and reliability of your gene expression analysis, so you should check them using methods such as gel electrophoresis, qPCR, or bioanalyzer.</span> </p> </div> </div> </div> </div> </div> <div class="column-article-segment-contributions md:column md:pb-[200px] md:w-1/2"> <div class="article-segment-contributions-header-container bg-color-canvas-tint md:ml-[-12px] md:pt-6 lg:ml-0 sticky-on-scroll mt-1 ">  <div class="add-your-perspective-section relative flex flex-row items-center " data-segment-urn="urn:li:articleSegment:(urn:li:linkedInArticle:7161790903102214144,7161790907422339072)" data-test-add-contribution> <code id="add_your_perspective_tracking_prefix" style="display: none"></code> <code id="isGuest" style="display: none"></code> <code id="showFuseErrorMessage" style="display: none"></code> <div class="mb-2"> <a 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You'll no longer see this contribution</p> </template> </li>  </ul>  </div> </div> <code id="contributionCopyLink" style="display: none"></code> <code id="contributionCopyFail" style="display: none"></code> <code id="contributionCopyUnsupported" style="display: none"></code> </div> </div> <div class="contribution__container left__border -mb-2 -mt-1 animate-show ml-0 pl-0 md:ml-[-41px] md:pl-[32px] pr-0 lg:ml-[-33px] lg:pl-[40px] lg:pr-1 pt-2 pb-4"> <div class="flex flex-col justify-between lg:flex-col"> <div class="flex items-center justify-between gap-1"> </div> </div> <div class="contribution__text-wrapper relative"> <p class="contribution__text mb-1 whitespace-pre-wrap pt-1 text-md leading-open text-color-text md:mb-0 md:pt-0 line-clamp-5 lg:text-lg" data-tracking-control-name="comment_text">I agree. Sample preparation is the first critical step. Extraction of RNA from biological samples needs to be done carefully under the hood so that it doesn’t get contaminated. Kits and reagents need to be separated. Once cDNA is ready, keep continuing the protocol for fragmentation, adapter ligation, etc. Make sure QC is done to check concentrations, size , etc. appropriate for sequencing breadth and depth.</p> <button class="contribution__see-more-button absolute bottom-0 right-0 hidden cursor-pointer border-0 bg-color-surface p-0 text-md leading-open text-color-text-secondary hover:text-color-action hover:underline lg:text-lg" data-tracking-control-name="comment_expand" data-feed-control="comment_expand" data-feed-action-type="expandContributionText" data-feed-action-category="EXPAND" role="button" type="button">…see more</button> </div>  <div class="contribution__social-action-bar relative right-1 flex flex-row items-center justify-start gap-1"> <div class="contribution-social-activity inline-flex items-center justify-start"> <code id="i18n_reaction_selection_state" style="display: none"></code> <div class="reactions-menu flex flex-row relative items-center justify-center select-none 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You can use different sequencing platforms, such as Illumina, PacBio, or Oxford Nanopore, depending on your budget, throughput, and research questions. You should also choose the appropriate sequencing depth, read length, and paired-end or single-end mode for your experiment. After sequencing, you should perform quality control of your raw data using tools such as FastQC, MultiQC, or Trim Galore. These tools can help you identify and remove low-quality reads, adapter contamination, or other artifacts that can affect your gene expression analysis.</span> </p> </div> </div> </div> </div> </div> <div class="column-article-segment-contributions md:column md:pb-[200px] md:w-1/2"> <div class="article-segment-contributions-header-container bg-color-canvas-tint md:ml-[-12px] md:pt-6 lg:ml-0 sticky-on-scroll mt-1 ">  <div class="add-your-perspective-section relative flex flex-row items-center " data-segment-urn="urn:li:articleSegment:(urn:li:linkedInArticle:7161790903102214144,7161790907426545664)" data-test-add-contribution> <code id="add_your_perspective_tracking_prefix" style="display: none"></code> <code id="isGuest" style="display: none"></code> <code id="showFuseErrorMessage" style="display: none"></code> <div class="mb-2"> <a href="https://www.linkedin.com/signup/cold-join?session_redirect=%2Fadvice%2F3%2Fhow-can-you-use-rna-seq-data-analyze-gene-rokle&trk=article-ssr-frontend-x-article" target="_self" data-tracking-control-name="add_contribution_cta" data-tracking-will-navigate data-test-link class="add-your-perspective-button btn-sm flex flex-col items-start justify-center lg:btn-md bg-color-surface-accent-tint-1 hover:bg-color-container-tertiary-hover " data-test-add-contribution-btn aria-describedby="content-urn_li_articleSegment__urn_li_linkedInArticle_7161790903102214144_7161790907426545664_" data-test-contribution-cta-link-guest role="button"> <div data-test-contribution-btn-text class="add-your-perspective-button-text text-left text-color-text-low-emphasis">Add your perspective</div> </a> </div> <div class="contribution-text-area-wrapper relative w-full hidden"> <code id="min_char_limit" style="display: none"></code> <code id="min_reply_char_limit" style="display: none"></code> <code id="max_char_limit" style="display: none"></code> <code id="i18n_contribution_post_success" style="display: none"></code> <code id="i18n_contribution_post_error" style="display: none"></code> <code id="i18n_contribution_edit_post_success" style="display: none"></code> <code id="i18n_contribution_edit_post_error" style="display: none"></code> <code id="i18n_try_refreshing_the_page" style="display: none"></code> <code id="i18n_characters" style="display: none"></code> <code id="i18n_characters_fraction" style="display: none"></code> <code id="i18n_contribution_post_rate_limit_error" style="display: none"></code> <code id="i18n_contribution_post_rate_limit_error_suggestion" style="display: none"></code> <div class="contribution-text-area-border ease-linear duration-300 mb-2 rounded-md border-1 border-solid border-[var(--color-input-container-border)] p-2 transition-all focus-within:shadow-border hover:shadow-border active:shadow-border-active md:px-3"> <textarea class="contribution-text-area leading-normal placeholder:text-color-input-hint box-border w-full resize-none border-0 bg-color-transparent p-0 text-md leading-open tracking-wide text-color-text outline-none placeholder:text-color-text-disabled lg:text-lg" placeholder="Some ways to get started: - 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Some tools (such as DESeq2) will do this for you, other tools (such as edgeR) required you to do this yourself. Failure to perform this step will result in much less significant adjusted p-values (or false discovery rates) after multiple correction testing. It is also important to consider any factors in your experiment (such a sample batch) that may influence gene expression, and ensure you control for these by including them in your design matrix</p> <button class="contribution__see-more-button absolute bottom-0 right-0 hidden cursor-pointer border-0 bg-color-surface p-0 text-md leading-open text-color-text-secondary hover:text-color-action hover:underline lg:text-lg" data-tracking-control-name="comment_expand" data-feed-control="comment_expand" data-feed-action-type="expandContributionText" data-feed-action-category="EXPAND" role="button" type="button">…see more</button> </div>  <div class="contribution__social-action-bar relative right-1 flex flex-row items-center justify-start gap-1"> <div class="contribution-social-activity inline-flex items-center justify-start"> <code id="i18n_reaction_selection_state" style="display: none"></code> <div class="reactions-menu flex flex-row relative items-center justify-center select-none 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data-entity-urn="urn:li:comment:(urn:li:articleSegment:(urn:li:linkedInArticle:7161790903102214144,7161790907439132672),7166468208332361728)" tabindex="0"> <img alt data-reaction-type="LIKE" data-delayed-url="https://static.licdn.com/aero-v1/sc/h/9sun4diznbhgyelaeryxy75ur" height="16px" width="16px"> <span aria-hidden="true" class="font-normal ml-0.5" data-test-id="social-actions__reaction-count"> 2 </span> </div> <code id="social-actions__reaction-image-APPRECIATION" style="display: none"></code>  <code id="social-actions__reaction-image-EMPATHY" style="display: none"></code> <code id="social-actions__reaction-image-ENTERTAINMENT" style="display: none"></code>  <code id="social-actions__reaction-image-INTEREST" style="display: none"></code> <code id="social-actions__reaction-image-LIKE" style="display: none"></code> <code id="social-actions__reaction-image-MAYBE" style="display: none"></code> <code id="social-actions__reaction-image-PRAISE" style="display: none"></code>  </div> </div>  </div>  </div>  </li> </ul>  </div> </div> </div> <code id="loginUrl" style="display: none"></code> <div class="article-segment" data-is-logged-in="false" data-thread-urn="urn:li:articleSegment:(urn:li:linkedInArticle:7161790903102214144,7161790907443326976)" data-test-id="article-segment" data-total-count="0" data-tracking-prefix="article-ssr-frontend-x-article" role="presentation"> <div class="md:row pb-6 md:flex"> <div class="column-article-segment-content md:column md:mb-8 md:mr-4 md:w-1/2 md:flex-1 md:pr-[88px]"> <div class="md:pt-6 sticky-on-scroll"> <div class="article-segment-content-container "> <div id="content-urn_li_articleSegment__urn_li_linkedInArticle_7161790903102214144_7161790907443326976_"> <div class="article-main__content" data-test-id="publishing-text-block"> <h2 class="flex"> <a id="step-5:-interpret-and-visualize-the-results" href="#step-5:-interpret-and-visualize-the-results" class="dummy-anchor invisible absolute mt-[-100px]"></a> <span class="header-index pr-1.5">5</span> <span class>Step 5: Interpret and visualize the results</span> </h2> </div> <div class="article-main__content" data-test-id="publishing-text-block"> <p> <span class>The fifth step is to interpret and visualize the results of your differential expression analysis using tools such as R, Bioconductor, or Python. This step will allow you to explore the biological meaning and significance of your differentially expressed genes or transcripts, and communicate your findings to others. You can use various methods to interpret and visualize the results, such as gene ontology analysis, pathway analysis, gene set enrichment analysis, volcano plots, heatmaps, or clustering.</span> </p> </div> </div> </div> </div> </div> <div class="column-article-segment-contributions md:column md:pb-[200px] md:w-1/2"> <div class="article-segment-contributions-header-container bg-color-canvas-tint md:ml-[-12px] md:pt-6 lg:ml-0 sticky-on-scroll mt-1 ">  <div class="add-your-perspective-section relative flex flex-row items-center " data-segment-urn="urn:li:articleSegment:(urn:li:linkedInArticle:7161790903102214144,7161790907443326976)" data-test-add-contribution> <code id="add_your_perspective_tracking_prefix" style="display: none"></code> <code id="isGuest" style="display: none"></code> <code id="showFuseErrorMessage" style="display: none"></code> <div class="mb-2"> <a href="https://www.linkedin.com/signup/cold-join?session_redirect=%2Fadvice%2F3%2Fhow-can-you-use-rna-seq-data-analyze-gene-rokle&trk=article-ssr-frontend-x-article" target="_self" data-tracking-control-name="add_contribution_cta" data-tracking-will-navigate data-test-link class="add-your-perspective-button btn-sm flex flex-col items-start justify-center lg:btn-md bg-color-surface-accent-tint-1 hover:bg-color-container-tertiary-hover " data-test-add-contribution-btn aria-describedby="content-urn_li_articleSegment__urn_li_linkedInArticle_7161790903102214144_7161790907443326976_" data-test-contribution-cta-link-guest role="button"> <div data-test-contribution-btn-text class="add-your-perspective-button-text text-left text-color-text-low-emphasis">Add your perspective</div> </a> </div> <div class="contribution-text-area-wrapper relative w-full hidden"> <code id="min_char_limit" style="display: none"></code> <code id="min_reply_char_limit" style="display: none"></code> <code id="max_char_limit" style="display: none"></code> <code id="i18n_contribution_post_success" style="display: none"></code> <code id="i18n_contribution_post_error" style="display: none"></code> <code id="i18n_contribution_edit_post_success" style="display: none"></code> <code id="i18n_contribution_edit_post_error" style="display: none"></code> <code id="i18n_try_refreshing_the_page" style="display: none"></code> <code id="i18n_characters" style="display: none"></code> <code id="i18n_characters_fraction" style="display: none"></code> <code id="i18n_contribution_post_rate_limit_error" style="display: none"></code> <code id="i18n_contribution_post_rate_limit_error_suggestion" style="display: none"></code> <div class="contribution-text-area-border ease-linear duration-300 mb-2 rounded-md border-1 border-solid border-[var(--color-input-container-border)] p-2 transition-all focus-within:shadow-border hover:shadow-border active:shadow-border-active md:px-3"> <textarea class="contribution-text-area leading-normal placeholder:text-color-input-hint box-border w-full resize-none border-0 bg-color-transparent p-0 text-md leading-open tracking-wide text-color-text outline-none placeholder:text-color-text-disabled lg:text-lg" placeholder="Some ways to get started: - One time at work… - In my experience… - One thing I’ve found helpful…" rows="4" data-segment-urn="urn:li:articleSegment:(urn:li:linkedInArticle:7161790903102214144,7161790907443326976)" aria-errormessage="error-urn:li:articleSegment:(urn:li:linkedInArticle:7161790903102214144,7161790907443326976)" data-feed-control="comment_box" data-feed-action-category="EXPAND" data-feed-action-type="expandContributionBox"></textarea> <div class="contribution-action-bar flex w-full items-center pt-2"> <span class="contribution-helper-text grow text-left text-xs text-color-text-secondary lg:text-sm" role="alert"> <span class="contribution-character-count"></span> <span class="contribution-error-message hidden text-color-signal-negative" id="error-urn:li:articleSegment:(urn:li:linkedInArticle:7161790903102214144,7161790907443326976)"> Help others by sharing more (125 characters min.) </span> </span> <button class="contribution-cancel-button ease-linear duration-300 btn-sm btn-tertiary mr-1 transition-all lg:btn-md" data-tracking-control-name="cancel_contribution"> Cancel </button> <button disabled="true" aria-disabled="true" class="submit-contribution-button ease-linear duration-300 btn-sm btn-primary transition-all lg:btn-md" data-tracking-control-name="comment_post" data-feed-control="comment_post" data-feed-action-category="COMMENT" data-feed-action-type="submitContribution"> <div class="loader flex"> <div class="loader__container mb-2 overflow-hidden"> <icon class="loader__icon inline-block loader__icon--default text-color-progress-loading" data-delayed-url="https://static.licdn.com/aero-v1/sc/h/ddi43qwelxeqjxdd45pe3fvs1" data-svg-class-name="loader__icon-svg--x-small fill-currentColor h-[20px] min-h-[20px] w-[20px] min-w-[20px]"></icon> </div> </div> <span class="submit-contribution-button-text">Add</span> <span class="save-contribution-changes-button-text">Save</span> </button> </div> </div> </div> </div> </div> <ul class="contribution-list contribution-list-guest">  </ul>  </div> </div> </div> <code id="loginUrl" style="display: none"></code> <div class="article-segment" data-is-logged-in="false" data-thread-urn="urn:li:articleSegment:(urn:li:linkedInArticle:7161790903102214144,7161790907447517184)" data-test-id="article-segment" data-total-count="0" data-tracking-prefix="article-ssr-frontend-x-article" role="presentation"> <div class="md:row pb-6 md:flex"> <div class="column-article-segment-content md:column md:mb-8 md:mr-4 md:w-1/2 md:flex-1 md:pr-[88px]"> <div class="md:pt-6 sticky-on-scroll"> <div class="article-segment-content-container "> <div id="content-urn_li_articleSegment__urn_li_linkedInArticle_7161790903102214144_7161790907447517184_"> <div class="article-main__content" data-test-id="publishing-text-block"> <h2 class="flex"> <a id="step-6:-validate-and-extend-the-results" href="#step-6:-validate-and-extend-the-results" class="dummy-anchor invisible absolute mt-[-100px]"></a> <span class="header-index pr-1.5">6</span> <span class>Step 6: Validate and extend the results</span> </h2> </div> <div class="article-main__content" data-test-id="publishing-text-block"> <p> <span class>The final step is to validate and extend the results of your gene expression analysis using other methods or data sources. This step will allow you to confirm the reliability and reproducibility of your results, and discover new insights or hypotheses. You can use various methods to validate and extend the results, such as RT-qPCR, western blot, RNA interference, CRISPR-Cas9, or single-cell RNA-seq.</span> </p> </div> </div> </div> </div> </div> <div class="column-article-segment-contributions md:column md:pb-[200px] md:w-1/2"> <div class="article-segment-contributions-header-container bg-color-canvas-tint md:ml-[-12px] md:pt-6 lg:ml-0 sticky-on-scroll mt-1 ">  <div class="add-your-perspective-section relative flex flex-row items-center " data-segment-urn="urn:li:articleSegment:(urn:li:linkedInArticle:7161790903102214144,7161790907447517184)" data-test-add-contribution> <code id="add_your_perspective_tracking_prefix" style="display: none"></code> <code id="isGuest" style="display: none"></code> <code id="showFuseErrorMessage" style="display: none"></code> <div class="mb-2"> <a href="https://www.linkedin.com/signup/cold-join?session_redirect=%2Fadvice%2F3%2Fhow-can-you-use-rna-seq-data-analyze-gene-rokle&trk=article-ssr-frontend-x-article" target="_self" data-tracking-control-name="add_contribution_cta" data-tracking-will-navigate data-test-link class="add-your-perspective-button btn-sm flex flex-col items-start justify-center lg:btn-md bg-color-surface-accent-tint-1 hover:bg-color-container-tertiary-hover " data-test-add-contribution-btn aria-describedby="content-urn_li_articleSegment__urn_li_linkedInArticle_7161790903102214144_7161790907447517184_" data-test-contribution-cta-link-guest role="button"> <div data-test-contribution-btn-text class="add-your-perspective-button-text text-left text-color-text-low-emphasis">Add your perspective</div> </a> </div> <div class="contribution-text-area-wrapper relative w-full hidden"> <code id="min_char_limit" style="display: none"></code> <code id="min_reply_char_limit" style="display: none"></code> <code id="max_char_limit" style="display: none"></code> <code id="i18n_contribution_post_success" style="display: none"></code> <code id="i18n_contribution_post_error" style="display: none"></code> <code id="i18n_contribution_edit_post_success" style="display: none"></code> <code id="i18n_contribution_edit_post_error" style="display: none"></code> <code id="i18n_try_refreshing_the_page" style="display: none"></code> <code id="i18n_characters" style="display: none"></code> <code id="i18n_characters_fraction" style="display: none"></code> <code id="i18n_contribution_post_rate_limit_error" style="display: none"></code> <code id="i18n_contribution_post_rate_limit_error_suggestion" style="display: none"></code> <div class="contribution-text-area-border ease-linear duration-300 mb-2 rounded-md border-1 border-solid border-[var(--color-input-container-border)] p-2 transition-all focus-within:shadow-border hover:shadow-border active:shadow-border-active md:px-3"> <textarea class="contribution-text-area leading-normal placeholder:text-color-input-hint box-border w-full resize-none border-0 bg-color-transparent p-0 text-md leading-open tracking-wide text-color-text outline-none placeholder:text-color-text-disabled lg:text-lg" placeholder="Some ways to get started: - One time at work… - In my experience… - One thing I’ve found helpful…" rows="4" data-segment-urn="urn:li:articleSegment:(urn:li:linkedInArticle:7161790903102214144,7161790907447517184)" aria-errormessage="error-urn:li:articleSegment:(urn:li:linkedInArticle:7161790903102214144,7161790907447517184)" data-feed-control="comment_box" data-feed-action-category="EXPAND" data-feed-action-type="expandContributionBox"></textarea> <div class="contribution-action-bar flex w-full items-center pt-2"> <span class="contribution-helper-text grow text-left text-xs text-color-text-secondary lg:text-sm" role="alert"> <span class="contribution-character-count"></span> <span class="contribution-error-message hidden text-color-signal-negative" id="error-urn:li:articleSegment:(urn:li:linkedInArticle:7161790903102214144,7161790907447517184)"> Help others by sharing more (125 characters min.) </span> </span> <button class="contribution-cancel-button ease-linear duration-300 btn-sm btn-tertiary mr-1 transition-all lg:btn-md" data-tracking-control-name="cancel_contribution"> Cancel </button> <button disabled="true" aria-disabled="true" class="submit-contribution-button ease-linear duration-300 btn-sm btn-primary transition-all lg:btn-md" data-tracking-control-name="comment_post" data-feed-control="comment_post" data-feed-action-category="COMMENT" data-feed-action-type="submitContribution"> <div class="loader flex"> <div class="loader__container mb-2 overflow-hidden"> <icon class="loader__icon inline-block loader__icon--default text-color-progress-loading" data-delayed-url="https://static.licdn.com/aero-v1/sc/h/ddi43qwelxeqjxdd45pe3fvs1" data-svg-class-name="loader__icon-svg--x-small fill-currentColor h-[20px] min-h-[20px] w-[20px] min-w-[20px]"></icon> </div> </div> <span class="submit-contribution-button-text">Add</span> <span class="save-contribution-changes-button-text">Save</span> </button> </div> </div> </div> </div> </div> <ul class="contribution-list contribution-list-guest">  </ul>  </div> </div> </div> <code id="loginUrl" style="display: none"></code> <div class="article-segment" data-is-logged-in="false" data-thread-urn="urn:li:articleSegment:(urn:li:linkedInArticle:7161790903102214144,7161790907455913984)" data-test-id="article-segment" data-total-count="1" data-tracking-prefix="article-ssr-frontend-x-article" role="presentation"> <div class="md:row pb-6 md:flex"> <div class="column-article-segment-content md:column md:mb-8 md:mr-4 md:w-1/2 md:flex-1 md:pr-[88px]"> <div class="md:pt-6 "> <div class="article-segment-content-container "> <div id="content-urn_li_articleSegment__urn_li_linkedInArticle_7161790903102214144_7161790907455913984_"> <div class="article-main__content" data-test-id="publishing-text-block"> <h2 class="flex"> <a id="here’s-what-else-to-consider" href="#here’s-what-else-to-consider" class="dummy-anchor invisible absolute mt-[-100px]"></a> <span class="header-index pr-1.5">7</span> <span class>Here’s what else to consider</span> </h2> </div> <div class="article-main__content" data-test-id="publishing-text-block"> <p> <span class>This is a space to share examples, stories, or insights that don’t fit into any of the previous sections. What else would you like to add?</span> </p> </div> </div> </div> </div> </div> <div class="column-article-segment-contributions md:column md:pb-[200px] md:w-1/2"> <div class="article-segment-contributions-header-container bg-color-canvas-tint md:ml-[-12px] md:pt-6 lg:ml-0 mt-1 ">  <div class="add-your-perspective-section relative flex flex-row items-center " data-segment-urn="urn:li:articleSegment:(urn:li:linkedInArticle:7161790903102214144,7161790907455913984)" data-test-add-contribution> <code id="add_your_perspective_tracking_prefix" style="display: none"></code> <code id="isGuest" style="display: none"></code> <code id="showFuseErrorMessage" style="display: none"></code> <div class="mb-2"> <a href="https://www.linkedin.com/signup/cold-join?session_redirect=%2Fadvice%2F3%2Fhow-can-you-use-rna-seq-data-analyze-gene-rokle&trk=article-ssr-frontend-x-article" target="_self" data-tracking-control-name="add_contribution_cta" data-tracking-will-navigate data-test-link 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How to Analyze Gene Expression with RNA-seq Data