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Homogenizing buffer

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Homogenizing Buffer is a solution designed to aid in the disruption and lysis of cells and tissues for the extraction of biomolecules, such as proteins, nucleic acids, and other cellular components. Its core function is to provide a controlled environment that facilitates the effective homogenization of samples, enabling the release and solubilization of the desired analytes.

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Lab products found in correlation

Quantigene 2.0 assay, by Thermo Fisher Scientific (9 mentions) Proteinase k, by Thermo Fisher Scientific (9 mentions) Rnalater, by Merck Group (7 mentions) M1000, by Tecan (6 mentions) Tissuelyser 2, by Qiagen (4 mentions) Collection microtubes, by Qiagen (2 mentions) Proteinase k, by Qiagen (1 mentions) M1000 luminometer, by Tecan (1 mentions)

10 protocols using homogenizing buffer

1

Quantifying mRNA Expression in Tissues

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At 1-week post-injection, mice were euthanized and perfused with PBS. Tissues were collected and stored in RNAlater (Sigma) at 4°C overnight. mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix). The 1.5-mm punches (three punches per tissue) were placed in QIAGEN Collection Microtubes holding 3-mm tungsten beads and lysed in 300 μl Homogenizing Buffer (Affymetrix) containing 0.2 mg/ml proteinase K (Invitrogen) using a QIAGEN TissueLyser II. Samples were then centrifuged at 1000 × g for 10 min and incubated for 1 h at 55° to 60°C. Lysates and diluted probe sets (mouse Htt, mouse Ppib or mouse Hprt) were added to the bDNA capture plate and signal was amplified and detected as described by Coles et al. (36 (link)). Luminescence was detected on a Tecan M1000 (Tecan, Morrisville, NC, USA).
Biscans A., Coles A., Haraszti R., Echeverria D., Hassler M., Osborn M, & Khvorova A. (2018). Diverse lipid conjugates for functional extra-hepatic siRNA delivery in vivo. Nucleic Acids Research, 47(3), 1082-1096.
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2

mRNA Quantification in Mouse Tissues

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At 1-week post-injection, mice were euthanized. Tissues were collected and stored in RNAlater (Sigma) at 4°C overnight. mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix). Briefly, 1.5-mm punches (3 punches per tissue) were placed in QIAGEN Collection Microtubes holding 3-mm tungsten beads and lysed in 300 μl Homogenizing Buffer (Affymetrix) containing 0.2 mg/ml proteinase K (Invitrogen) using a QIAGEN TissueLyser II. Samples were then centrifuged at 1,000 × g for 10 min and incubated for 1 h at 55° to 60°C. Lysates and diluted probe sets (mouse Htt, or mouse Hprt) were added to the bDNA capture plate and signal was amplified and detected as described by Coles et al. [29 (link)]. Luminescence was detected on a Tecan M1000 (Tecan, Morrisville, NC, USA).
Biscans A., Coles A., Echeverria D, & Khvorova A. (2019). The valency of fatty acid conjugates impacts siRNA pharmacokinetics, distribution, and efficacy in vivo. Journal of controlled release : official journal of the Controlled Release Society, 302, 116-125.
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3

Quantifying mRNA Expression in Tissues

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At 1-week post-injection, tissues were collected. mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix). Tissue punches were lysed in 300 μl Homogenizing Buffer (Affymetrix) containing 0.2 mg/ml proteinase K (Invitrogen). Diluted lysates and probe sets (mouse Htt, mouse Ppib, or mouse Hprt) were added to the bDNA capture plate and signal was amplified and detected as described by Coles et al. [36 (link)]. Luminescence was detected on a Tecan M1000 (Tecan, Morrisville, NC, USA).
, & Hutchinson T.A. (2001). Herons and the human side of medicine. CMAJ: Canadian Medical Association Journal, 164(9), 1326.
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4

Quantifying mRNA Levels in Tissues

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At 1-week post-injection, tissues were collected and stored in RNAlater (Sigma) at 4°C overnight. mRNA was then quantified using the QuantiGene 2.0 Assay (Affymetrix). Briefly, tissue punches were lysed in 300 μl Homogenizing Buffer (Affymetrix) containing 0.2 mg/ml proteinase K (Invitrogen). Diluted lysates and probe sets (mouse Htt, mouse Ppib, or mouse Hprt) were added to the bDNA capture plate and signal was amplified and detected as described by Coles et al. (39 (link)). Luminescence was detected on a Tecan M1000 (Tecan, Morrisville, NC, USA).
Biscans A., Caiazzi J., Davis S., McHugh N., Sousa J, & Khvorova A. (2020). The chemical structure and phosphorothioate content of hydrophobically modified siRNAs impact extrahepatic distribution and efficacy. Nucleic Acids Research, 48(14), 7665-7680.
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5

Quantifying mRNA Expression in Tissues

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Tissues were collected and stored in RNAlater (Sigma) at 4°C overnight. mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix). Tissue punches were lysed in 300 μL Homogenizing Buffer (Affymetrix) containing 0.2 mg/mL proteinase K (Invitrogen). Diluted lysates and probe sets (mouse Htt or mouse Hprt) were added to the bDNA capture plate and the signal was amplified and detected as described previously60 (link).
Yamada K., Hariharan V.N., Caiazzi J., Miller R., Furguson C., Sapp E., Fakih H., Tan Q., Yamada N., Furgal R.C., Paquette J., Bramato B., McHugh N., Summers A., Lochmann C., Godinho B.M., Hildebrand S., Echeverria D., Hassler M.R., Alterman J.F., DiFiglia M., Aronin N, & Khvorova A. (2023). Extended Nucleic Acid (exNA): A Novel, Biologically Compatible Backbone that Significantly Enhances Oligonucleotide Efficacy in vivo.. bioRxiv.
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6

Quantification of mRNA Expression

Cited 1 time
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Tissues were collected and stored in RNAlater (Sigma) at 4 °C overnight. mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix). Tissue punches were lysed in 300 μL Homogenizing Buffer (Affymetrix) containing 0.2 mg/mL proteinase K (Invitrogen). Diluted lysates and probe sets (mouse CD47 or mouse Hprt) were added to the bDNA capture plate and the signal was amplified and detected as described previously (59 (link)). Luminescence was detected on a Tecan M1000 (Tecan). Flow cytometry-based CD47 expression analysis was performed as described previously (48 (link)).
Hariharan V.N., Shin M., Chang C.W., O’Reilly D., Biscans A., Yamada K., Guo Z., Somasundaran M., Tang Q., Monopoli K., Krishnamurthy P.M., Devi G., McHugh N., Cooper D.A., Echeverria D., Cruz J., Chan I.L., Liu P., Lim S.Y., McConnell J., Singh S.P., Hildebrand S., Sousa J., Davis S.M., Kennedy Z., Ferguson C., Godinho B.M., Thillier Y., Caiazzi J., Ly S., Muhuri M., Kelly K., Humphries F., Cousineau A., Parsi K.M., Li Q., Wang Y., Maehr R., Gao G., Korkin D., McDougall W.M., Finberg R.W., Fitzgerald K.A., Wang J.P., Watts J.K, & Khvorova A. (2023). Divalent siRNAs are bioavailable in the lung and efficiently block SARS-CoV-2 infection. Proceedings of the National Academy of Sciences of the United States of America, 120(11), e2219523120.
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7

Quantifying mRNA Expression in Tissues

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Tissues were collected and stored in RNAlater (Sigma) at 4°C overnight. mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix). Tissue punches were lysed in 300 μL Homogenizing Buffer (Affymetrix) containing 0.2 mg/mL proteinase K (Invitrogen). Diluted lysates and probe sets (mouse Htt or mouse Hprt) were added to the bDNA capture plate and the signal was amplified and detected as described previously60 (link).
Hariharan V.N., Caiazzi J., Miller R., Ferguson C., Sapp E., Fakih H., Tang Q., Yamada N., Furgal R., Paquette J., Bramato B., McHugh N., Summers A., Lochmann C., Godinho B., Hildebrand S., Echeverria D., Hassler M., Alterman J., DiFiglia M., Aronin N., Khvorova A, & Yamada K. (2023). Extended Nucleic Acid (exNA): A Novel, Biologically Compatible Backbone that Significantly Enhances Oligonucleotide Efficacy in vivo. Research Square.
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8

Quantitative mRNA Measurement Methodology

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mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix) as described previously (8 (link)). Briefly, cells were lysed in 250 μl-diluted lysis mixture with Proteinase K (Affymetrix) for 30 min at 55°C prior to mRNA quantification. Tissue punches (∼5 mg) were homogenized in 300 μl of Homogenizing Buffer (Affymetrix) with Proteinase K in 96-well plate format using a TissueLyser II (Qiagen). This method is described in detail in Coles et al (11 (link)).
Osborn M.F., Coles A.H., Biscans A., Haraszti R.A., Roux L., Davis S., Ly S., Echeverria D., Hassler M.R., Godinho B.M., Nikan M, & Khvorova A. (2018). Hydrophobicity drives the systemic distribution of lipid-conjugated siRNAs via lipid transport pathways. Nucleic Acids Research, 47(3), 1070-1081.
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9

Quantification of mRNA Levels in Tissues

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At 1 week post-injection or 1 month post-injection, tissues were collected and stored in RNAlater (Sigma) at 4°C overnight. mRNA was quantified using the QuantiGene 2.0 Assay (Affymetrix). Tissue punches were lysed in 300 μL Homogenizing Buffer (Affymetrix) containing 0.2 mg/mL Proteinase K (Invitrogen). Diluted lysates and probe sets (mouse Htt, mouse Mstn, or mouse Hprt) were added to the bDNA capture plate, and the signal was amplified and detected as described by Coles et al.74 (link) Luminescence was detected on a Tecan M1000 (Tecan, Morrisville, NC, USA).
Biscans A., Caiazzi J., McHugh N., Hariharan V., Muhuri M, & Khvorova A. (2020). Docosanoic acid conjugation to siRNA enables functional and safe delivery to skeletal and cardiac muscles. Molecular Therapy, 29(4), 1382-1394.
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10

Quantification of mRNA in Cells and Tissue

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mRNA was quantified from both cells and tissue punches using the QuantiGene® 2.0 DNA Assay (Affymetrix) exactly as described previously1 , 48 (link). Probe sets (Mouse Htt or Hprt) were diluted and used according to the manufacturer’s recommended protocol. Briefly, tissue punches (5 mg) were homogenized in 300 µl of Homogenizing Buffer (Affymetrix) containing 2 µg/µl proteinase K in 96-well plate format on a QIAGEN TissueLyser II, and 40 µl of each lysate was added to a bDNA capture plate. Htt or Hprt probe sets (60 µL) were added to each well of the capture plate for a final volume of 100 µL. Signal was amplified according to the Affymetrix protocol. Luminescence was detected on a Tecan M1000 luminometer.
Nikan M., Osborn M.F., Coles A.H., Biscans A., Godinho B.M., Haraszti R.A., Sapp E., Echeverria D., DiFiglia M., Aronin N, & Khvorova A. (2017). Synthesis and Evaluation of Parenchymal Retention and Efficacy of a Metabolically Stable, O-phosphocholine-N-docosahexaenoyl-L-serine siRNA Conjugate in Mouse Brain. Bioconjugate chemistry, 28(6), 1758-1766.
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