Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Primers for qPCR validation in non-model RNA-Seq

    Hey guys,

    Once we got differentially expressed (DE) genes using RNA-Seq, we need to do qPCR validation.

    1.For a non-model plant, among the DE genes, maybe most of them are unknown. If you pick some known and unknown genes/transcripts to do qPCR validation, how do you design primers for those unknown (maybe novel) genes/transcripts? (qPCR primers span introns)
    2.Generally, how many genes should be selected as candidate genes?


    Any suggestions would be greatly appreciated.
    Thanks.

    Sincerely,
    lzsph

  • #2
    1. You could design a number of PCR primers at discrete intervals (0, 150, 300, 450bp) along the transcript, then test in pairwise combinations using genomic DNA vs cDNA as template. Any pair that produces bands of different sizes (gDNA > cDNA) spans an intron (you can confirm by Sanger sequencing the PCR products).

    2. It really depends upon the goal of your qPCR experiment. Remember to include a couple of non-differential genes as controls, though.

    Comment


    • #3
      Originally posted by HESmith View Post
      1. You could design a number of PCR primers at discrete intervals (0, 150, 300, 450bp) along the transcript, then test in pairwise combinations using genomic DNA vs cDNA as template. Any pair that produces bands of different sizes (gDNA > cDNA) spans an intron (you can confirm by Sanger sequencing the PCR products).

      2. It really depends upon the goal of your qPCR experiment. Remember to include a couple of non-differential genes as controls, though.
      Got it! Thank you HESmith.

      Comment

      Latest Articles

      Collapse

      • seqadmin
        Strategies for Sequencing Challenging Samples
        by seqadmin


        Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
        03-22-2024, 06:39 AM
      • seqadmin
        Techniques and Challenges in Conservation Genomics
        by seqadmin



        The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

        Avian Conservation
        Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
        03-08-2024, 10:41 AM

      ad_right_rmr

      Collapse

      News

      Collapse

      Topics Statistics Last Post
      Started by seqadmin, Yesterday, 06:37 PM
      0 responses
      10 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, Yesterday, 06:07 PM
      0 responses
      9 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 03-22-2024, 10:03 AM
      0 responses
      51 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 03-21-2024, 07:32 AM
      0 responses
      67 views
      0 likes
      Last Post seqadmin  
      Working...
      X