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What's all this nonsense about contamination?

Discussion in 'XMRV Research and Replication Studies' started by jace, Mar 1, 2011.

  1. jace

    jace Off the fence

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    How PCR was first calibrated to find XMRV by Urisman et al [1]

    First off, the Virochip technique was used to screen RNA samples isolated from prostate tumours. Notice that XMRV was not isolated by PCR and there was no attempt to target viral DNA and we are not talking about isolating XMRV from blood.

    The RNA was amplified and purified into something called polyadenylated RNA. This is free of any DNA or anything else like histones. It is viral RNA (if present) and human RNA. This was then converted into cDNA and the fragments were isolated using something called a microarray.

    This next bit is a bit technical but its meaning is crucial: Briefly, amplified nucleic acid from the tumour tissue, which hybridized to viral microarray oligonucleotides, was eluted from two specific spots.

    The eluted DNA was re-amplified, and plasmid libraries constructed from this material were screened by colony hybridization using the spots' oligonucleotides as probes. The array oligonucleotides used in this case derived from the LTR region of murine type C retrovirus (MTCR) and spleen focus-forming virus (SFFV) [2].

    The largest recovered fragment was 415 nt in length, and had 96% nt identity to the LTR region of MTCR, notice the multiple rounds of amplification. This is not PCR and the viral nucleic acid was picked up by primers complimentary for a LTR region.

    Now most people realise that the XMRV genome is made up of GAG POL and ENV. This however is not the whole story. The genome has two identical regions of RNA at each side of the GAG POL ENV sequences. Now about 8% of our DNA is made up of endogenous retroviral sequences.

    The reason that we are not all dead is because these babies cant replicate. Some of these are classed as MLV endogenous retroviruses. The key thing to remember that these ERVs have deletions and mutations which mean that they can't make the proteins needed to make virus particles. Now bearing this in mind I present the following diagram
    [​IMG]
    Now the upside down mountains are deletions and hence the HERVs can't code for proteins of the length needed. Bearing in mind that the original samples only contained RNA where would a non human ERV have come from? As XMRV can code for the full compliment of proteins needed to construct a virion it cannot by definition be a ERV. It is amazing how the Coffin cabal have missed this point.

    Naked viral RNA cannot survive in a cellular environment and hence cannot be a contaminant. If there was contaminant DNA then the detection method used would not have selected it and it would not have entered the experimental environment.

    As a final point in this instalment the authors in this study developed a PCR assay which could detect viral sequences in patient tissue known to be infected using the original micro array technique. They were able to do this by constructing primers complimentary to the gag sequences isolated by the other method.

    They did this by adjusting the cycling conditions until they could detect the virus in DNA made from the RNA they isolated from the patients tissue. They had the advantage of knowing that the virus was present, and could change the parameter of the PCR reaction until they found it. They also used a form of PCR called reverse transcription PCR. That is for another time. Note the presence of mouse DNA is an impossibility as the process began with polyadenylated RNA!

    1] Urisman A, Molinaro RJ, Fischer N, Plummer SJ, Casey G, Klein EA, Malathi K, Magi-Galluzzi C, Tubbs RR, Ganem D, et al: Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant. PLoS Pathog 2006, 2:e25.

    2] Clark SP, Mak TW (1983) Complete nucleotide sequence of an infectious clone of Friend spleen focus-forming provirus: gp55 is an envelope fusion glycoprotein. Proc Natl Acad Sci U S A 80: 5037–5041.
  2. lansbergen

    lansbergen Senior Member

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    http://en.wikipedia.org/wiki/Polyadenylation

    Polyadenylation is the addition of a poly(A) tail to an RNA molecule. The poly(A) tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA which only has adenine bases.
  3. alex3619

    alex3619 Senior Member

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    Hi jace, I really enjoyed your post as usual. However, I have two questions, that I am not up to answering right this minute, but I think are important. Are we certain the specific cell lines now being blamed for contamination only contain inactivated XMRV DNA? Second, is the XMRV found in for example prostate cancer samples really inactivated or activated viral DNA - how can we be sure? A mixture of activated and deactivated is possible too. (Sorry I might not be making sense, I am not able to sleep at the moment.)

    Let me address this partially. Your first post on this thread identifies the viral RNA as the original isolated sequence, and by inference it is from a life virus, not an ERV. This does not prove that the prostate cancer cell lines don't have live virus and so could be a source of contamination. How hard have they looked for live virion RNA in these cell lines?

    Of course, the capacity to clone and infect macaques directly implies this is a real virus and so capable of infecting humans. This implies that some people may carry live XMRV, a point which has not been disproven. I still think that if contamination is occurring, the most likely source is infected humans. This implies there is a background rate. This further implies that the so called zero zero studies should have found some XMRV. Therefore there is something wrong with the testing.

    Is there something wrong with my reasoning?

    Bye
    Alex
  4. SilverbladeTE

    SilverbladeTE Senior Member

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    Somewhere near Glasgow, Scotland
    yes, you are quite wrong, because you are sane and logical!
    this makes you automatically an "EPIC FAIL" in the world of "Captain Weasel and the World of Tomorrow" :D

    in case anyone misconstrues:
    I believe Alex is 100% correct and am supporting him in my own weird way...it's just that being logical, compassionate or having the slightest common sense what so ever, is completely at odds with the retarded, inhumane lingam-licking system we live in! ;)
  5. natasa778

    natasa778 Senior Member

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    Logical, who cares about logical?

    Would Cort or Kurt care to teach Alter a lesson?
  6. Wonko

    Wonko Senior Member

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    Just found this thread but I like it

    (Sorry not qualified to add tech stuff - I'm a bit thick these days)
  7. Doogle

    Doogle Senior Member

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    natasa778, that is very persuasive where was the Alter statement presented?
  8. WillowJ

    WillowJ Senior Member

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    at the blood work group. he also said the contamination guys were arguing from "anecdotal" examples.

    Some of those transcritps are great to read. Alter and Mikovits operate like a cross-examination team in a courtroom. :thumbsup:
  9. WillowJ

    WillowJ Senior Member

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    here we go: official blood products advisory meeting transcript

    Dr. Alter again:
  10. jace

    jace Off the fence

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    Hi Alex,
    You may guess I'm in a different time-zone than you! It does mean I have time to do other things while you all sleep...;)

    What it actually means is that it could have come from virions, eukaryotic or prokaryotic messenger RNA. They were not using prostate cell lines, the tissue was taken directly from patients with prostate cancer. Whatever was there was a MLV virus because the cells reacted with a monoclonal antibody.

    The point is that any viral DNA would not have been picked up because the samples were polyadenylated RNA. What was investigated was the presence of viral RNA. They did not use prostate cell lines of any kind in the experiment. FISH detected viral nucleic acid within and outside the nucleus. This can only be viral m RNA or cDNA (genomic viral RNA can't enter the nucleus of a host.

    Whatever was in the tissue was replicating and making MLV specific proteins. No Type C HERV (human endogenous retrovirus) can manufacture SU proteins. It can't have been a HERV. There is also no known endogenous or exogenous MLV in humans, mice or any other mammal with that SU sequence. This is some times described as a VRA and a VRB region.

    To answer the specific point about whether the DNA is active or inactive, MLV viruses engage in post integrational latentcy and can indeed become silent. In this case however viral nucleic acid was observed both within the nucleus and without. This coupled with the presence of MLV SU proteins means that the proviral DNA was being transcribed. The IHC test is only positive if a replicating virus is involved.

    Hope this helps.
  11. alex3619

    alex3619 Senior Member

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    Hi Jace, thank you, very illuminating. Now all I have to do is remember it all. I am really struggling with memory issues. Bye, Alex

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