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How not to find Viral RNA The Dutch approach

Discussion in 'XMRV Research and Replication Studies' started by Gerwyn, Feb 28, 2010.

  1. Gerwyn

    Gerwyn Guest

    I am not familiar with the exact techniques for obtaining viral RNA from whole blood especially from blood frozen for such a long time so I thought I,d look up the "receipe".I dont think the Dutch trrialists were very good Cooks

    Collection and Storage of Human Blood Cells for mRNA Expression Profiling: A 15-Month Stability Study
    Jean-Brice Marteau, Steve Mohr, Michle Pfister and Sophie Visvikis-Siesta
    Institut National de la Sant et de la Recherche Mdicale, U525 Equipe 4, Facult de Pharmacie, Nancy, France;
    aaddress correspondence to this author at: INSERM U525 Equipe 4, 30 rue Lionnois, 54000 Nancy, France; fax 33-3-83321322, e-mail Sophie.Visvikis-Siest@nancy.inserm.fr



    The next bit shows that if you take GREAT care the RNA isolated from blood samples is OK after 15 MONTHS not 15 YEARS as they tried in the dutch study
    .Surely a virologist who had done is or her homework would know this


    After informed consent was obtained from 12 unrelated adult volunteers, whole blood (10 mL) was collected by standardized venipuncture in EDTA (EDTA-blood; n = 12) and heparin (heparin-blood; n = 12) tubes (VacutainerTM; Becton Dickinson) and processed for PBMC preparation. Briefly, blood samples were homogenized with 10 mL of Hanks Balanced Salt Solution, Modified (Sigma-Aldrich). PBMCs were prepared by Ficoll density-gradient centrifugation (Ficoll-PaqueTM PLUS; Amersham) at 300g for 30 min at 20 C. The ring of high-density PBMCs was isolated and washed twice in 50 mL of Hanks buffer. After cell survival was determined with the trypan-blue exclusion test, the PBMC concentration was normalized to 106 cells/mL in Hanks buffer. PBMC populations were evaluated by microscopic observation after May–Grunwald–Giemsa staining. After centrifugation for 6 min at 700g (4 C), 200 L of cell lysis buffer was immediately added to the PBMC pellet. Four different commercially available buffers were tested (n = 24 for each buffer, including 12 EDTA-blood and 12 heparin-blood samples): RNA InstaPure [Eurogentec (E)], lysis/binding buffer from the MagNA Pure LC RNA Isolation Kit I [Roche Diagnostics (R)], buffer RTL from the QIAamp RNA Blood Mini Kit [Qiagen (Q)], and RNA lysis buffer from the StrataPrep Total RNA Miniprep Kit [Stratagene (S)]. The PBMC lysates were used to extract total RNA immediately (t0) or after storage at –80 C for 3, 5, 10, and 15 months (t3, t5, t10, and t15, respectively).



    This next bit shows how you measure whether your extracted RNA is still competent or totally destroyed by the extraction process.RNA is MUCH more unstable than DNA.Note they didn,t chuck some pig virus RNA into the sample and hope for the best like the Dutch Study did..With great care taken the RNA was OK after 15 MONTHS NOT 15 YEARS like the samples in the dutch study

    Total RNA quality and quantity were assessed in 2 ways.

    In the first method, we estimated the RNA concentration by ultraviolet absorbance at 260 nm (1 absorbance unit at 260 nm = 40 ng/L RNA) and the RNA purity by measuring the ratio of absorbance at 260 nm and 280 nm (1.8 < A260/A280 < 2.1 for pure RNA). Total RNA was run on 1% agarose gels to check size and integrity.

    In the second method, RNA from each sample was assessed for integrity by a 2-step reverse transcription-PCR assay for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; NM_002046). cDNA synthesis was performed at 37 C for 60 min with 80 ng of extracted RNA, 0.25 g of oligo(dT)15 primer, and 200 U of Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega).


    Our studies demonstrate that PBMC RNA is stable over time (up to 15 months) when blood samples are collected into EDTA tubes and when PBMCs are stored at –80 C in buffer E. We provide here a simple assay for rapid, efficient, and standardized banking of PBMCs, a key step in large gene expression studies. To our knowledge, this is the first comprehensive report on RNA stability over 15 months.

    The next bit is how to get viral rna

    J Clin Microbiol. 1998 February; 36(2): 493–498. PMCID: PMC104566
    Copyright 1998, American Society for Microbiology
    Stability of Human Immunodeficiency Virus RNA in Blood Specimens as Measured by a Commercial PCR-Based Assay
    Kimberley Sebire, Kate McGavin, Sally Land, Tracey Middleton, and Chris Birch*

    This next section shows you what you need to do to locate RNA from superthug aids let alone its weeny cousin XMRV.Note how you get at the RNA in the first place.And how you have to extract the blood You need to use specific enzymes specific primers Tested after 7 to ten days not 15 **** years

    Blood specimens were collected from HIV-seropositive individuals attending outpatient clinics at several hospitals and private clinics in Melbourne, These specimens arrived at the testing laboratory within 6 h after being drawn, the time limit for separation of plasma recommended by the manufacturer.

    Except where otherwise stated, 8 to 10 ml of whole blood was collected in tubes containing 1.3% (wt/vol) acid citrate dextrose (ACD) (Greiner, Labortechnik) and transported to the laboratory by courier.

    When used, EDTA tubes were obtained from Greiner, Labortechnik. Plasma was collected by centrifugation at 400 g for 10 min at room temperature (RT; 21C) and stored at −70C for between 5 and 10 days prior to being

    t

    The Amplicor HIV-1 Monitor assay was used according to the manufacturer’s instructions in all tests requiring HIV RNA quantitation.

    Briefly, target RNA was prepared by guanidine thiocyanate lysis of HIV virions in plasma, followed by isopropanol precipitation. The enzyme rTth DNA polymerase was used to reverse transcribe the RNA into cDNA and to amplify DNA by PCR.

    The biotinylated products of amplification were diluted in fivefold steps and hybridized to target-specific oligonucleotide probes immobilized in the wells of a microtiter plate.

    This procedure was followed by enzyme immunoassay-based colorimetric detection of the bound products.

    Quantitation was accomplished by the inclusion of a known concentration of synthetic RNA (containing the same primer binding sites as the target HIV RNA but with a unique probe sequence), which was reverse transcribed and coamplified with the target RNA.




    HIV was isolated from patient peripheral blood mononuclear cells (PBMC) by coculture with phytohemagglutinin-stimulated donor PBMC (17).

    Patient PBMC were separated from 8 to 10 ml of ACD-treated blood by density gradient centrifugation. Patient and donor PBMC were cocultured in a one-to-one ratio in a medium containing RPMI 1640 (ICN Biomedicals, Inc., Costa Mesa, Calif.), 10% heat-inactivated fetal calf serum (Cytosystems, Castle Hill, Australia), and 10% recombinant human interleukin-2 (Boehringer Mannheim Australia Pty Ltd., Castle Hill, Australia).

    Cocultures were maintained at 37C in 5% CO2 for up to 28 days.


    During this time, fresh medium was added twice every 7 days, and fresh donor PBMC were added once every 7 days. A detectable increase in p24 antigen levels in supernatant fluid, measured with a commercially available enzyme immunoassay kit (Vironstika HIV-1 Antigen Microelisa system; Organon Teknika Corp., Durham, N.C.), was considered to indicate active virus replication.

    This method is well known but totally ignored by the dutch
    notice even with spuperthug the only sure way of getting at the RNA is the lysis of whole virion particles which are REPLICATIVE
     
  2. bettine

    bettine

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    Hi Gerwyn,
    I am trying very hard to understand what you are saying, and believe me for a layperson it is a very difficult subject.
    But one of the things you seem to be saying is that you cannot use blood that is 15 years old. But didn't they use stored 'old' blood in the Science study?
    And do you think you could explain what you wrote in such a way that a lay person can understand?
     
  3. Lesley

    Lesley Senior Member

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    The WPI Bio Bank contains blood collected from 2006-2009. The samples for the Science study came from the Bio Bank.

    All the studies have used stored samples, but I believe the Dutch samples were by far the oldest.
     
  4. George

    George Guest

    bettine

    The short answer is more or less as follows (grins)

    You can use stored blood samples to find viral RNA if you know that you are going to use the blood to look for RNA

    1. and you have the blood drawn correctly.
    2. and you use the correct tubes. (different tubes have different stuff in the bottoms of them like detergents and preservatives)
    3. and you freeze the blood correctly. (poor freezing can ruin samples)
    4. and when you unfreeze the blood you do it correctly.
    5. and when you look for the viral partial you apply the correct amplifiers. (Cause any virus is going to need amplification after 15 years of being frozen and if it doesn't you better run cause that's one really tough virus and it will likely eat you when you unfreeze it!)
    6. and then you culture it to get it going ( pipe, slippers, nice seat by the fire and a couple of lady viruses to get reproducing.)
    Then you get to put it in the PCR, and check it against a know sample of the virus. If you got virus then you get these pretty little white bands on the black cards and they match up.

    Now let clarify the WPI samples. Yes, in the verrrrrry begining the WPI used some of the frozen samples of the people who had Lymphoma and using a viro chip they got a few hits on this new virus. That was enough to get them to design the Science Study. Here they used blood from patients that was relatively fresh. (Kind of like when people sent their blood to VIP it had to get there in 24 hours.) The blood used in the Science Study was less than 2 years old and had been properly handled, stored and was not frozen.

    People get mixed up I think. Because WPI did look at some frozen blood samples at one point and talked about that (the Lymphoma patients from Dr. Peterson's) then people think all the blood for the Science Study must have been frozen old blood. That's incorrect. If you look at the Science supplement you'll find that the blood used for the study did not come from Dr. Peterson at all but from clinics around the US that work with CFS patients. Turns out that some of the patients being treated in these clinics were from different places around the world.

    The WPI used those clinics because there "had" been outbreaks in those areas and so doctors had become CFS specialist because of the need. Because it is so difficult to find doctors it turns out that patients from all over the US and the World migrated to those few clinics in order to get a doctor with some understanding of the illness.

    Hope that helps my brain is a little fried right now (grins)
     
  5. oerganix

    oerganix Senior Member

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    Thank you George! Masterful as usual. When Gerwyn speaks that other language, you are there to interpret and we are grateful to you both.
     
  6. Gerwyn

    Gerwyn Guest


    hi Bettine sorry in geek mode

    The science says that you can get working RNA in blood taken and processed VERY carefully if you freeze it for 15months at the longest

    This crew tried to get working RNA in samples not treated at all carefully after 15 years.

    Despite no one ever trying this before they decided not to use the tests to see if the RNA was actually working and not destroyed

    They instead threw in some fresh RNA from an endogenous Retrovirus and claimed that their test worked
    If you do RT PCR on fresh porcine virus it will work ! and produce porcine c DNA which of course is not XMRV .They would have lots of pretty bands to look at.But there would not be XMRV if it was not there inthe first place

    The longest time that T and B cells have been shown to survive after freezing is two years---This is where XMRV lives.For XMRV RNA to show up the B and T cells have to be replicating. No one knows if these cells can replicate after 15 years in a very deep freeze.But again they did not bother to check.

    There is a recepie for these things--They were trying to make a chicken curry but used a recipie for making chicken soup
     
  7. George

    George Guest

    Dang Gerwyn, NOW that was MASTERFUL!
     
  8. mezombie

    mezombie Senior Member

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    East Coast city, USA
    Thanks to two of my favorite geeks, Gerwyn and George, for explaining this so that even my scrambled brain could understand it!

    Important stuff to know when we see new studies like the Dutch one!

    :Green hat: Hats off to you both!
     
  9. usedtobeperkytina

    usedtobeperkytina Senior Member

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    Clay, Alabama
    By George, I think we've got it.

    I know DeFrietas' discovery was doubted, even by her own later attempts, but I remember clearly that the virus she found COULD NOT BE FROZEN AT ALL! She said freezing corrupts the genetic material. This was the main issue with the CDC study trying to confirm her claims. CDC froze the samples, sometimes for convenience because the researcher was on vacation at the time, etc.

    And remember, yes, WPI did declare post-article that they found it in one of Peterson's 1980s patients. But remember, they were using four different methods. If the Dutch had used all four of these methods, maybe they would have found it in the frozen blood. But it is not expected they would have tried all four methods. But, if they are going to declare their study casts doubt on WPI, then they sure should have followed those methods.

    Now, was the explanation of samples from within last two years and handling of samples in the original Science Journal article? I don't read science speak, so someone tell me. Was it in the supplemental. I am getting two different versions here. Vernon says not enough, but others say its there if you bother to read it. Won't do me any good to read it. So was the info about when blood drawn, handling of the blood, amplification, etc. etc. in the original published info or supplemental? If so, then shame on you researchers in Europe. If it wasn't, then sorry you wasted your time and money over a lack of information.

    Tina
     
  10. kolowesi

    kolowesi Senior Member

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    Central Texas
    old and frozen blood

    Hi Ladybug,

    I just re-listened to Dr. Peterson's presentation. I believe he said that the plasma from a very old frozen sample contained virion which could infect cell lines. Plasma not containing actual cells. Perhaps this is the explanation?

    I have been very confused about all this, so I'm not sure, even though I did just listen to it. You know how it is:Retro tongue:

    Kelly
     
  11. Gerwyn

    Gerwyn Guest

    they did find at least oneI believe.in the Dutch study they were trying to Find viral RNA direct from frozen blood.it is quite different and you are quite right the dutch study did not use plasma but went for the PMBC route assuming there were whole virions about with RNA to work on without checking any thing in any way
     
  12. Gerwyn

    Gerwyn Guest

    The correct way to collect and store blood for RNA processing

    The DUTCH study is not even close The processes for blood taking and storage are critical if you hope to get viable RNA

    RNA Sample Collection and Submission Guidelines
    Services | RNA Specimen Processing Overview | RNA Extraction FAQ | Sample Collection and Submission Guidelines | Methods and Data Delivery | Scheduling and Shipping

    Please contact GPCL personnel to clarify procedures before beginning sample collection.

    Sample Collection
    In order to maximize the probability of success in your goals we recommend that you follow some basic procedures during sample collection to ensure RNA quality.

    Tissue Collection
    Tissue must be protected from degradation and cross contamination throughout the collection process. Gloves should be worn at all times and any instrument used in removing or handling the tissue must be well cleaned and free of even residual material from any other subject or tissue specimen. If the tissue needs to be split it should be rapidly cut with a clean blade. The tissue should be quickly deposited into an appropriate clean container and frozen in liquid nitrogen. If prompt storage in liquid nitrogen is not possible

    , specimens may be preserved in RNALater (Ambion).

    Specimens can be minced to <0.5 cm as needed with a clean blade then quickly submerged in 5-10 volumes of RNALater solution

    . Per RNALater manufacturer's specifications, the RNA is protected from degradation for 24 hours at 37C, 1 week at 25C, or 1

    month at 4C. For archival storage the samples should be incubated at 4C overnight then transferred to -20 or -80C.

    The RNALater MUST be removed prior to freezing!

    This is a particularly desirable method for small biopsies taken in a clinic setting where liquid nitrogen is inaccessible or inconvenient or for shipping specimens overseas where there is risk of delays.

    Tissue Storage Containers
    Tissue specimens brought to the Core Laboratories should be put into a cryovial with a screw-top lid. If specimens will not fit in a cryovial, please contact the GPCL for best collection and submission practices. The preferred handling and preservation method for most samples is snap freezing in liquid nitrogen with subsequent transportation on dry ice and long term storage at -80C.

    Blood Collection
    Blood for RNA extraction should be drawn into a PaxGene (Qiagen) or Tempus (Applied Biosystems) tube.

    It is CRITICAL FOR RNA quality and yield that tubes be thoroughly mixed by inversion at the time of collection, that a full tube of blood be taken and that nothing is placed over the black fill mark on the manufacturer's label.

    Please be sure the collecting personnel have been thoroughly briefed in collection procedures.


    Tubes can be refrigerated for up to one week or frozen at -80C for up to a YEAR before being delivered to the GPCL for processing.

    For further details see Shipping/Scheduling instructions.

    Blood Tube Labels
    All sample tubes must be labeled with the following information in order to be processed: the Study Name, PI Name, Study Reference ID, and the Date Collected. Any other pertinent information regarding the sample must be noted, i.e. infectious status (if known). Sample labels must NOT contain any information revealing the identity of clinical subjects. Please use adherent labels that do not appreciably enlarge the diameter of the tubes so that they can be placed into a centrifuge as needed. See notification section in Shipping/Scheduling for further details.

    Storage Containers for Mail Delivery


    Blood samples must be packaged in containers that conform to postal regulations for biohazardous materials and mailed OVERNIGHT to the GPCL Specimen Processing Core Facility for Next day delivery.


    Frozen tubes must be shipped in dry ice.

    Cells
    Cell culture source cells may be provided by the Investigator for RNA isolation. Minimum handling is recommended to avoid changing the expression profile of living cells. Cells grown in suspension should be pelleted, media completely removed and snap frozen. No wash is necessary. Adherent cells should be lysed with TRIzol (Invitrogen) and the lysate submitted for processing. Please contact GPCL personnel for clarification on any of these procedures.

    Sample Log Sheet
    A Specimen Processing drop off form must be received with all samples. Studies involving multiple drop offs over a period of months may use a project specific sample log sheet as arranged during the project initiation meeting. Standard drop off forms are available at: http://www.genetics.pitt.edu/forms/

    Labels for Containers
    All sample tubes must be labeled with the following information in order to be processed: the Study Name, PI Name, Study Reference ID, Date collected. Sample labels must NOT contain any information revealing the identity of clinical subjects. All tubes/containers should be labeled with printed labels as laboratory reagents frequently dissolve most marker inks.

    Drop-off
    Samples may also be dropped off at one of our sample drop off locations, 3343 Forbes Ave Room 303, and S534A Scaife Hall or 2.15 Hillman Cancer Center Research Pavilion. Frozen tissue samples should be delivered to 3343 Forbes Avenue on dry ice to avoid potential thawing.
     
  13. kurt

    kurt Senior Member

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    USA.Earth
    The Dutch study goes into some detail about the RNA because that was a more difficult process, but they did a total extraction, meaning they also looked at DNA. The DNA finding is more solid. Gerwyn is correct that there was more risk with the RNA extraction, they did not fully prove viability of the RNA, however it was present along with the DNA, which probably surprised them. Anyway my point is that the DNA was evaluated and that is the critical part of the study. Sorry, but the RNA problem does not shoot down this study, only the RNA portion of the finding which in fact might have still been viable, they just did not prove that adequately to be totally convincing.

    If this study were ONLY of the RNA, then the point Gerwyn is making would have discredited the entire study. However, the fact that they did a total extraction means they looked at DNA. They also looked at copy DNA or cDNA, but that was a separate step.
     
  14. Gerwyn

    Gerwyn Guest

    Kurt they isolated DNA They did not know whether they had degraded rna or not but they clearly state in their study that there were no problems with it.Isolating DNA is one thing isolating proviral DNA in non replicating cells quite another
     

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