Announcement

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

  • Strange peak in total RNA extractions from brain tissue

    We have extracted total RNA from frozen brain tissue. To extract RNA, we homogenized 30-50mg tissue with Trizol then added chloroform for phase separation and lastly did a buffer exchange using the miRNeasy kit. We did not perform DNase treatment. We are seeing a large peak at around 100-150nt from the bioanalyzer for all 40 brain samples we used regardless of RNA integrity numbers (see attachment). In fact most RINs are high. Has anyone else seen this? Is this degraded RNA? If it was degraded RNA, wouldn't it be a smear? Or is it possible it is DNA?
    Attached Files

  • #2
    Those are your small RNAs. tRNA, 5S, 5.8S, etc. Many RNA prep kits do not efficiently isolate them, but Trizol can -- depending on your post-extraction concentration/purificiation method. "miRNeasy" would stand for "micro RNA easy". So one would expect lots of small RNA there.

    In some samples isolated with Trizol we see small RNA peaks so high that the BioAnalyzer software balks and refuses to give a RIN until you suppress certain errors it generates.

    --
    Phillip

    Comment


    • #3
      Is this peak specific to brain or do you see it across tissue types when using Trizol?

      I wasn't certain this peak was attributed to small RNA species like pri-miRNA or tRNA because I didn't think they wouldn't this abundant. Also, pri-miRNAs are 70nt and tRNA are in the 70-90nt range. Do you think it's mostly 5S, 5.8S rRNA?

      Thanks for the help Phillip!

      -Andy

      Comment


      • #4
        Well, I can't rule out your peak being something else -- something brain-specific, or even degradation products. But to me it just looks like normal RNA with lots of small RNA in it. However it is a little strange that there is nothing from 25-100 nt. Is it possible that this is something like the "anti-miRNA-enriched" fraction of your RNA?

        --
        Phillip

        Comment


        • #5
          I know there is miRNA present bc I've done qPCR on specific targets. I'm pretty sure you nailed with it by saying it is 5S, 5.8S and degraded RNA.

          Thanks for your help

          -Andy

          Comment


          • #6
            Bioanalyzer band shift 150 -> 100 nt

            Dear all, we encountered shift at the similar band size. We isolated total RNA from neuronal cell culture using combination of QIAzol Lysis reagent (QIAGEN) and Direct-zol RNA MiniPrep (Zymo Research). Quality of the isolated RNA was checked on Expirion (Bio-Rad Laboratories) (direct competitor of Agilent Bioanalyzer). Under control conditions, we have observed relatively defined double-band at ~ 150 nt (green). Upon an experimental treatment the double-band weakened and new broad band with most intensive part at ~ 100nt appeared (orange, see image attached). It was consistent in all cases when treatment was used. Do you have an idea what class of RNAs could be responsible for the shift and why could this shift occur? Thank you for response!
            Attached Files

            Comment


            • #7
              Interesting that I got the exact peaks / bands from insect fat body tissue using almost the exact same experimental set-up (zymo micro kit & Experion). I have virtually eliminated that this might be degradation, but can't figure out what else it might be other than a high amount of material at the 5S/5.8S/tRNA/miRNA region. Did anyone else get any conclusive answers?
              Attached Files

              Comment


              • #8
                Have you tried depleting the rRNA using Ribo-Zero or RNAse H methods and seeing if or how many of the small RNAs remain or are eliminated using Bioanalyzer?

                Comment


                • #9
                  Originally posted by Olaf Blue View Post
                  Have you tried depleting the rRNA using Ribo-Zero or RNAse H methods and seeing if or how many of the small RNAs remain or are eliminated using Bioanalyzer?
                  No and it may be too late for me to do that. I am actually trying to retain the small RNAs and I don't think the presence of the ribosomes is necessarily a problem for my downstream goals. I just didn't expect the peak to be so high! I may need to eliminate the possibility that incomplete lysis of my tissue may be responsible for any of the height of the peak at this region and not RNA degradation for some other reason, which I am reasonably sure it is not. If it is not incomplete lysis, and degradation isn't seen near the larger ribosomal subunits (which they are not), then it may be a tissue-specific phenomenon.

                  I am trying to find a source that explains "Insufficient lysis is indicated by accumulation of small RNA around the 5S rRNA peak."

                  I appreciate anyone's thoughts!

                  Comment


                  • #10
                    So, correct me if I'm inferring the wrong thing, but the figure from the paper you link to is saying that incomplete lysis results in some of the RNA fractionating with debris that is removed from the RNA prep?

                    Do you think you got sufficient lysis? Insects often have cleavage sites in their large ribosomal sub-unit RNAs that result in cleavage products the same, or nearly the same length as the small ribosomal sub-unit RNA. We don't think twice if we see this result from insect RNA.

                    But you seemed concerned about differences in the yields of small RNA products. (Or, what I presume are small RNA products. You don't provide sizing information for the bands.) Unfortunately gels only give you so much information. Unless someone who has run a lot of gels on RNA obtained from the fat bodies of insects happens by the forum, you are unlikely to get actionable answers.

                    --
                    Phillip

                    Comment


                    • #11
                      Originally posted by pmiguel View Post
                      So, correct me if I'm inferring the wrong thing, but the figure from the paper you link to is saying that incomplete lysis results in some of the RNA fractionating with debris that is removed from the RNA prep?


                      Does "insufficient lysis" mean cells that are only partially lysed such that only the small RNAs are released and the rest is washed away with the cells? My original interpretation was that, perhaps, some of the cells were recalcitrant due to the nature of the tissue and, thus, took longer to break apart and were subject to endogenous RNases for longer periods of time?

                      Also, yes, well aware of the insect 18S/28S single peak.

                      Thanks.

                      Comment


                      • #12
                        Originally posted by eingabe_ausgabe View Post
                        http://www.nature.com/nprot/journal/...16.090_F2.html

                        Does "insufficient lysis" mean cells that are only partially lysed such that only the small RNAs are released and the rest is washed away with the cells? My original interpretation was that, perhaps, some of the cells were recalcitrant due to the nature of the tissue and, thus, took longer to break apart and were subject to endogenous RNases for longer periods of time?

                        Also, yes, well aware of the insect 18S/28S single peak.

                        Thanks.
                        Yes, your interpretation is more likely.

                        --
                        Phillip

                        Comment

                        Latest Articles

                        Collapse

                        • seqadmin
                          Multiomics Techniques Advancing Disease Research
                          by seqadmin


                          New and advanced multiomics tools and technologies have opened new avenues of research and markedly enhanced various disciplines such as disease research and precision medicine1. The practice of merging diverse data from various ‘omes increasingly provides a more holistic understanding of biological systems. As Maddison Masaeli, Co-Founder and CEO at Deepcell, aptly noted, “You can't explain biology in its complex form with one modality.”

                          A major leap in the field has
                          ...
                          02-08-2024, 06:33 AM
                        • seqadmin
                          The 3D Genome: New Technologies and Emerging Insights
                          by seqadmin


                          The study of three-dimensional (3D) genomics explores the spatial structure of genomes and their role in processes like gene expression and DNA replication. By employing innovative technologies, researchers can study these arrangements to discover their role in various biological processes. Scientists continue to find new ways in which the organization of DNA is involved in processes like development1 and disease2.

                          Basic Organization and Structure
                          Understanding...
                          01-22-2024, 03:25 PM

                        ad_right_rmr

                        Collapse

                        News

                        Collapse

                        Topics Statistics Last Post
                        Started by seqadmin, Yesterday, 08:57 AM
                        0 responses
                        12 views
                        0 likes
                        Last Post seqadmin  
                        Started by seqadmin, 02-14-2024, 09:19 AM
                        0 responses
                        43 views
                        0 likes
                        Last Post seqadmin  
                        Started by seqadmin, 02-12-2024, 03:37 PM
                        0 responses
                        410 views
                        0 likes
                        Last Post seqadmin  
                        Started by seqadmin, 02-09-2024, 03:36 PM
                        0 responses
                        649 views
                        0 likes
                        Last Post seqadmin  
                        Working...
                        X