Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • qPCR Validation of Single Sample

    I have a question regarding qPCR validation of some RNA-Seq data I am working with. We are working with a population that has never been characterized using RNA-Seq and due to sample availability our first run is a single sample of a pool RNA from multiple individuals. Due to it only being a single sample I am not sure how to report qPCR validation data. I am running 10 or so genes that span low to high FPKM from our expression data and should get a consistent pattern in terms of expression levels on qPCR and then will be sequencing the qPCR amplicons. However due to it only being on sample I see no way to use a housekeeping gene. Is it possible to somehow just take Ct values and report them in a manner that shows consistency with FPKM values for those genes from the seq data? Our next objective is comparing two different samples, I understand we could use housekeeping genes there but I am a little stuck on how to report this initial single sample global characterization study. Any help would be appreciated.

  • #2
    Hi ccard28

    So it seems your question carries the answer already.
    Basically let me remind you: housekeeping genes have one sole purpose: to allow relative expression comparison between samples at a globla scale. The only thing signifying a HK gene is that it is (presumably) not differentially expressed in the compared samples.
    So back to your one sample: I guess you want to confirm that several individual amplicons have a different level of representation in the sample. So to have an reference point to compare against you can choose any amplicon (since it doesnt have to stable across samples), or simply use the geometric mean of all the amplicons tested.
    And yes you could also simply present the Cts, only that is graphically not so intuitive because it doesnt really illuminate fold differences.
    A little hint for when you start comparing across samples: if you dont know any HK genes yet (?) the simplest way is to compare the seq data across all samples, searching for a statistically NOT diff expressed target. If its stable across your sample population, it is by definition a HK gene for these samples. -Off cause you need to include enough samples to have statiscial relevance, but guess you would anyway.

    Comment

    Latest Articles

    Collapse

    • seqadmin
      Exploring the Dynamics of the Tumor Microenvironment
      by seqadmin




      The complexity of cancer is clearly demonstrated in the diverse ecosystem of the tumor microenvironment (TME). The TME is made up of numerous cell types and its development begins with the changes that happen during oncogenesis. “Genomic mutations, copy number changes, epigenetic alterations, and alternative gene expression occur to varying degrees within the affected tumor cells,” explained Andrea O’Hara, Ph.D., Strategic Technical Specialist at Azenta. “As...
      07-08-2024, 03:19 PM
    • seqadmin
      Exploring Human Diversity Through Large-Scale Omics
      by seqadmin


      In 2003, researchers from the Human Genome Project (HGP) announced the most comprehensive genome to date1. Although the genome wasn’t fully completed until nearly 20 years later2, numerous large-scale projects, such as the International HapMap Project and 1000 Genomes Project, continued the HGP's work, capturing extensive variation and genomic diversity within humans. Recently, newer initiatives have significantly increased in scale and expanded beyond genomics, offering a more detailed...
      06-25-2024, 06:43 AM

    ad_right_rmr

    Collapse

    News

    Collapse

    Topics Statistics Last Post
    Started by seqadmin, 07-10-2024, 07:30 AM
    0 responses
    30 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 07-03-2024, 09:45 AM
    0 responses
    202 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 07-03-2024, 08:54 AM
    0 responses
    212 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 07-02-2024, 03:00 PM
    0 responses
    194 views
    0 likes
    Last Post seqadmin  
    Working...
    X