G
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
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
