Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • 260/230 and RNA-seq library prep

    Hi,
    Does anyone have experience of low 260/230 values for RNA impacting adversely on Illumina truseq total RNA library prep for RNA-seq? I have good 260/280 values and adequate quantities of RNA but low 260/230s. I have heard various opinions about the utility of 260/230 ratios but wondered what other experiences were? Thanks!

  • #2
    Originally posted by TAR76 View Post
    Hi,
    Does anyone have experience of low 260/230 values for RNA impacting adversely on Illumina truseq total RNA library prep for RNA-seq? I have good 260/280 values and adequate quantities of RNA but low 260/230s. I have heard various opinions about the utility of 260/230 ratios but wondered what other experiences were? Thanks!
    My opinion is that since you are probably using a Nanodrop that does all the work of producing an entire spectrum for you, why not just look at that rather than use a ratio metric designed back when you had to manually change wavelengths on most spectrophotometers?

    If you did, you might have a chance of identifying the contaminating substance by its UV absorbance spectrum.

    If you are willing to consider the spectrum, check out my thread on this subject:

    Techniques and protocol discussions on sample preparation, library generation, methods and ideas


    The short answer is that the most common culprits for causing your A230 to skew higher are:

    (1) Acetic acid and salts thereof.
    (2) Guanidine -- HCl and Isothiocyanate salts have different spectra
    (3) Betamercaptoethanol.

    Note that I don't include phenol! Phenol has a higher absorbance at A260 than at A230, so it can't be responsible for decreasing your 260/230 ratio. Oh, unless you are using Tris-acetate buffer to equilibrate your phenol. But then it is the acetate, not the phenol decreasing your 260/230 ratio.

    --
    Phillip

    Comment


    • #3
      Yes, we have seen that low 260/230 ratios negatively affects the TruSeq total RNA prep. When we experience low ratios, we always do a wash on a MinElute filter to improve purity.

      Comment


      • #4
        Originally posted by LTJensen View Post
        Yes, we have seen that low 260/230 ratios negatively affects the TruSeq total RNA prep. When we experience low ratios, we always do a wash on a MinElute filter to improve purity.
        Ah, a ritual of purification. I was in a lab once that had a shrine to the "oligod". Lab members would burn pieces of nitrocellulose there before an important molecular experiment.

        Here is something to think about -- what about all the "contaminants" that don't absorb UV? How are you detecting those?

        --
        Phillip

        Comment


        • #5
          I see your point pmiguel, nevertheless the ritual worked for us... When ratios are fine, we never have problems with that particular protocol.

          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, 03-27-2024, 06:37 PM
          0 responses
          15 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-27-2024, 06:07 PM
          0 responses
          13 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-22-2024, 10:03 AM
          0 responses
          55 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-21-2024, 07:32 AM
          0 responses
          70 views
          0 likes
          Last Post seqadmin  
          Working...
          X