Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Is there an upper limit for RNA input

    We have been having issues with our RNA from FFPE when submitting for sequencing. We are using the Illumina kit RS-122-2201, TruSeq Stranded Total RNA RboZroHMN/Mse/Rat with ribo depletion.

    Our samples are degraded so we have decided to use either no fragmentation or minimal fragmentation (Covaris) to start with.

    We are also considering using up to 750 ng of input RNA.

    I'm concerned that we may be using too much to start with and that can be problematic. We have a CORE facility that makes our libraries, so I'm not 100% familiar with the protocol for library construction - just a rough idea.

    If I don't need to use this much material, I would love to be able to save some for other downstream testing.

    Thank you.

  • #2
    Up to 1ug RNA can be used with this kit and it has instructions on how to fragment degraded RNA. Covaris should not be used for fragmenting RNA for this kit.

    Comment


    • #3
      What should be used? I know they are running RIN and DV200. Illumina has provided them with guidelines on how long to fragment on the Covarius based on the DV200. I know there is an enzymatic (tagment?) option, but again, they are going off recommendation by Illumina.

      Comment


      • #4
        The kit user guide recommends QC by Bioanalyzer and it has several RNA profiles along with recommended fragmentation protocol for each. DV200 is used for RNA enrichment protocols such as RNA access and not for the kit you have mentioned.

        Tagmentation is used for DNA libraries and it cannot be used directly on RNA.

        Comment


        • #5
          The stand-alone Ribo-Zero kits are to be used with 100 ng up to 5 ug of total RNA. In the past, Epicentre/Illumina's Scriptseq RNA-Seq kits with Ribo-Zero have been used for rRNA depletion and sequencing. No fragmentation is done.

          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
          49 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