Rna dna stabilization reagent for blood bone marrow

Manufactured by Roche

Sourced in Germany

The RNA/DNA Stabilization Reagent for Blood/Bone Marrow is a product designed to stabilize and preserve RNA and DNA samples derived from blood or bone marrow. It is intended to maintain the integrity of nucleic acids during collection, storage, and transportation.

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Lab products found in correlation

Magna pure lc rna isolation kit high performance kit, by Roche (2 mentions) Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions) Cfx 96 real time pcr system detector, by Bio-Rad (1 mentions) Mircury lna rt kit, by Qiagen (1 mentions) Moloney murine leukemia virus m mlv reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions) Simpliamp, by Thermo Fisher Scientific (1 mentions) L glutamine, by Merck Group (1 mentions) Mrna isolation kit for blood bone marrow, by Roche (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Ionomycin, by Merck Group (1 mentions) First strand cdna synthesis kit, by Roche (1 mentions) Magna pure lc rna isolation kit high performance, by Roche (1 mentions) Magna pure lc 2.0 instrument, by Roche (1 mentions)

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RNA Stabilization Reagent for Blood/Bone Marrow

5 protocols using rna dna stabilization reagent for blood bone marrow

Differential Expression of miRNAs in TB

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To analyze the expression of DE miRNAs (miR-16-5p, miR-199-3p (detection of both miR-199a-3p and miR-199b-3p), miR-374c-5p, miR-6886-3p, and miR-6856-3p), blood samples after MTB-specific peptide stimulation were treated using 500 µL of RNA/DNA stabilization reagent for blood/bone marrow (Roche Diagnostics, Mannheim, Germany). Subsequently, a MagNA Pure LC RNA Isolation Kit-High Performance kit (Roche Diagnostics) was used to extract RNA according to manufacturer’s instructions [30 (link)].
Next, complementary DNA (cDNA) of miR-16-5p, miR-199a-3p, miR-199b-3p, miR-374c-5p, miR-6886-3p, and miR-6856-3p was synthesized using the miRCURY LNA RT Kit (Qiagen) following manufacturer’s protocols. The temperature profile for cDNA synthesis reaction was as follows: 42 °C for 60 min and 95 °C for 5 min.
Quantitative reverse transcriptase (qRT)-PCR was performed using the miRCURY LNA miRNA PCR Assay [31 (link)]. PCR cycling was performed as follows: 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 s and 56 °C for 1 min. qRT-PCR were performed on the CFX96 Real-time PCR System Detector (Bio-Rad, Hercules, CA, USA). Samples were run in duplicate for each experiment. To monitor contamination of the reagents, a negative control was included for each primer pair. Data were analyzed using the comparative ΔC_T method (2^−ΔCT), with RNU44 as an endogenous control [32 (link)].

Kim J., Park H., Park S.B., Lee E.J., Je M.A., Ahn E., Sim B., Lee J., Jin H., Lee K.E., Cho S.N., Kang Y.A., Lee H., Kim S, & Kim J. (2022). Identification of MicroRNAs as Potential Blood-Based Biomarkers for Diagnosis and Therapeutic Monitoring of Active Tuberculosis. Diagnostics, 12(2), 369.

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Analyzing Cytokine Profiles in TB

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In order to analyze relative mRNA expressions of IFN-γ, TNF-α, IL-2R, CXCL9, IP-10, CXCL11, CCL11, GM-CSF, and TNFR after MTB-specific antigen stimulation, blood cell pellets were treated using 500 μL of RNA/DNA stabilization reagent for Blood/Bone marrow (Roche Diagnostics, Mannheim, Germany). Then, MagNA Pure LC RNA Isolation Kit-High Performance kit (Roche Diagnostics) was used for RNA isolation according to the manufacturer’s protocols [10 (link)]. The concentration and purity of extracted total RNA were measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the samples were stored at −80 °C until further use.
Next, complementary DNA (cDNA) was synthesized using a Moloney murine leukemia virus (M-MLV) reverse transcriptase kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocols. All reactions were performed using a SimpliAmp (Life Technologies, Carlsbad, CA, USA) thermal cycler.

Park J.Y., Park S.B., Park H., Kim J., Kim Y.N, & Kim S. (2021). Cytokine and Chemokine mRNA Expressions after Mycobacterium tuberculosis-Specific Antigen Stimulation in Whole Blood from Hemodialysis Patients with Latent Tuberculosis Infection. Diagnostics, 11(4), 595.

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Blood-based Mycobacterial Infection Assay

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One milliliter of blood, diluted 10-fold in HEPES-buffered Roswell Park Memorial Institute 1640 medium (Sigma-Aldrich, Missouri) supplemented with L-glutamine (Sigma-Aldrich), was cultured with 4 × 10⁵ colony-forming units of M. tuberculosis H37Rv or Mycobacterium bovis bacillus Calmette-Guerin strain Glaxo-Evans, corresponding to a multiplicity of infection of 1 bacillus to 1 monocyte, assuming 4 × 10⁵ monocytes/mL of peripheral blood [19 ]. After 6 days, supernatants were removed, cells were lysed in 10 mL of RNA/DNA Stabilization Reagent for Blood/Bone Marrow (Roche Applied Science, Basel, Switzerland), and specimens were frozen at −80°C.

Cliff J.M., Cho J.E., Lee J.S., Ronacher K., King E.C., van Helden P., Walzl G, & Dockrell H.M. (2015). Excessive Cytolytic Responses Predict Tuberculosis Relapse After Apparently Successful Treatment. The Journal of Infectious Diseases, 213(3), 485-495.

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Pharmacodynamic Monitoring Protocol

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The blood samples used for pharmacodynamic monitoring were obtained in parallel with the blood samples for pharmacokinetic monitoring and were analyzed at Vall d’Hebron Research Institute.
To do so, blood samples were collected in BD Vacutainer^® lithium heparin tubes. One milliliter of heparinized blood was stimulated with 1 mL of complete RPMI 1640 (Gibco) enriched with 100 ng/mL of phorbol 12-myristate 13-acetate (PMA) and 5 µg/mL of ionomycin (Sigma Aldrich, Saint Louis, MO) for three hours at 37°C and 5% CO₂. Then, 10 mL of RNA/DNA Stabilization Reagent for Blood/Bone Marrow (Roche, Mannheim, Germany) were added to lyse cells and stabilize nucleic acids, according to the manufacturer’s instructions. Samples were stored at -20°C for a maximum of a year.
mRNA was isolated using an mRNA Isolation Kit for Blood/Bone Marrow (Roche, Mannheim, Germany) following the manufacturer’s instructions. It was then stored at -20°C for a week or less, or at -80°C indefinitely.
Reverse transcription was performed with 8.2 µL of mRNA in a thermocycler using avian myeloblastosis virus reverse transcriptase and oligo (dT) as a primer (First Strand cDNA synthesis kit Roche, Mannheim, Germany) following the manufacturer’s instructions. The resulting cDNA was stored concentrated at -20°C until further use.

Boada-Pérez M., Ruiz de Miguel V., Erro M., Ussetti P., Aguilar M., Castejón R., Rosado S., Escobar-Fornieles R., Revilla-López E., Bravo C., Sáez-Giménez B., Zapata-Ortega M., Villena-Ortiz Y., Vima-Bofarull J., Monforte V, & Gómez-Ollés S. (2024). Pharmacodynamic monitoring by residual gene expression of the nuclear factor of activated T cell-regulated genes in lung transplant recipients and its correlation with tacrolimus blood levels. Frontiers in Immunology, 15, 1382459.

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Antigen-Stimulated Whole Blood RNA Extraction

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Antigen-stimulated whole blood cell pellets were treated with RNA/DNA Stabilization Reagent for Blood/Bone Marrow (Roche Diagnostics, Mannheim, Germany) and stored at À80 C until used for total RNA isolation. The MagNA Pure LC 2.0 instrument and the MagNA Pure LC RNA Isolation Kit-High Performance (Roche Diagnostics), which is a magnetic beadebased method, was used for total RNA extraction from antigen-stimulated whole blood cells. The M-MLV Reverse Transcriptase kit and random hexamers (Invitrogen, Carlsbad, CA) were used for synthesizing cDNA according to the manufacturer's recommendations. Briefly, 10 mL of extracted total RNA was added to a master mixture containing 1.0 mL 10 mmol/L dNTP mix, 0.25 mg random hexamers, and 5.0 mL diethyl-pyrocarbonateetreated water. The reaction mixture was incubated at 65 C for 5 minutes and quickly chilled on ice. Subsequently, a mixture of 4.0 mL 5 Â First-Strand Buffer, 2.0 mL 0.1 mol/L dithiothreitol, and 1.0 mL M-MLV reverse transcriptase was added to the previous reaction mixture and the cDNA synthesis reaction was performed for 10 minutes at 25 C, 50 minutes at 37 C, and 15 minutes at 70 C.

Kim S., Lee H., Kim H., Kim Y., Cho J.E., Jin H., Kim D.Y., Ha S.J., Kang Y.A., Cho S.N, & Lee H. (2015). Diagnostic performance of a cytokine and IFN-γ-induced chemokine mRNA assay after Mycobacterium tuberculosis-specific antigen stimulation in whole blood from infected individuals. The Journal of molecular diagnostics : JMD, 17(1).

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