Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • low 260/230 ratios of rRNA depleted and purified plant sample

    I am planning to construct libraries for RNAseq from ribosomal RNA depleted plant RNA. The total RNA samples (145ng/ul) were treated with RiboZero kits (illumina) and purified. After the RiboZero treatment, i quantified the RNA samples on Nanodrop, and noticed that the concentration of sample decreased to 53.7 (this was expected), however the 260/280 ratios was 1.78 (previously 1.8) and 260/230 ratios was 0.97( previously 1.7). I am concerned that these ratios would affect the downstream processes. Any ideas why these ratios could have changed? or suggestions on how I can improve these ratios? Any help would be appreciated

    PS: RNA sample volume is 8 ul and purification method was ethanol/sodium acetate incubation.

    Thank you

  • #2
    rRNA makes up more than 80% of total RNA prep. If your input volume also was 8 ul then I would suspect that rRNA depletion has been less successful. You should loos at least 80% of input mass.

    low 260/230 ratio could be due to residual sodium acetate from precipitation step or ethanol from wash. This can be corrected by re-precipitation and clean up more carefully but you will loos some RNA. Effect of low 260/230 will depend on the volume of downstream reaction and extent of contaminant.

    Comment

    Latest Articles

    Collapse

    • seqadmin
      Current Approaches to Protein Sequencing
      by seqadmin


      Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...
      04-04-2024, 04:25 PM
    • 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

    ad_right_rmr

    Collapse

    News

    Collapse

    Topics Statistics Last Post
    Started by seqadmin, 04-11-2024, 12:08 PM
    0 responses
    18 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 04-10-2024, 10:19 PM
    0 responses
    22 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 04-10-2024, 09:21 AM
    0 responses
    17 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 04-04-2024, 09:00 AM
    0 responses
    49 views
    0 likes
    Last Post seqadmin  
    Working...
    X