Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Effects of total RNA fragmentation on small RNAs

    Hello,
    I want to sequence enriched RNA (it will include both mRNAs and bacterial small non-coding RNAs (<200 nt)). I would like to fragment long RNAs to ~100-200 nt fragments before library preparation however I am afraid that small RNAs will be fragmented too and I will lose it after cutting required length RNAs from the gel (I am planning to cut 150-350 bp fragments already with adapters). My fragmentation buffer is 10 mM Tris-HCl (pH 7.4) and 10 mM ZnCl2, reaction conditions: 75 °C for 2.5 min.
    RNAs I want to sequence are not abundant - every loss of RNA sample should be avoided!
    Maybe someone has experience with the fragmentation of total RNA? Does it affect small RNAs, is it severely fragmented? Would it be better for me to prepare different libraries - one for small RNAs, another - for long RNAs?
    Thank you for your time and answer!

  • #2
    I assume chemical fragmentation will affect the small fragments to the same degree as the big ones.
    If you have an opportunity to sonicate samples? Small fragments are pretty safe from shearing.

    Another way to achieve that might be size selection (SPRI-beads) and separate fragmentation of the large fragments. Obviously, this will entail losses and add complexity.

    You might want to have a look at the Biorad Sequoia and Diagenode D-plex library prep kits for both short and long RNAs.

    Comment

    Latest Articles

    Collapse

    • seqadmin
      Essential Discoveries and Tools in Epitranscriptomics
      by seqadmin


      The field of epigenetics has traditionally concentrated more on DNA and how changes like methylation and phosphorylation of histones impact gene expression and regulation. However, our increased understanding of RNA modifications and their importance in cellular processes has led to a rise in epitranscriptomics research. “Epitranscriptomics brings together the concepts of epigenetics and gene expression,” explained Adrien Leger, PhD, Principal Research Scientist on Modified Bases...
      Yesterday, 07:01 AM
    • 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

    ad_right_rmr

    Collapse

    News

    Collapse

    Topics Statistics Last Post
    Started by seqadmin, 04-11-2024, 12:08 PM
    0 responses
    39 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 04-10-2024, 10:19 PM
    0 responses
    41 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 04-10-2024, 09:21 AM
    0 responses
    35 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 04-04-2024, 09:00 AM
    0 responses
    55 views
    0 likes
    Last Post seqadmin  
    Working...
    X