TRI Insta-Pure

Details for Product No. ABIN3188322, Supplier: Log in to see
Application
RNA Extraction (REx), Sample Preparation (Prep)
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Purpose RNA Insta-Pure is a single-step method for the isolation of total RNA from tissues, cells, bacteria, plants and yeasts.
Characteristics The entire procedure takes 1-2 hours and yields undergraded RNA that is free of protein and DNA contaminations.
This single reagent allows very high RNA purification yield and purity, even for tissues and cells containing high amounts of RNases.
The purified RNA contains the entire spectrum of RNA strands, including small (4-5 S) RNAs.
The ease of the RNA Insta-Pure method combined with excellent recovery from small quantities of tissues or cells makes this product especially suitable for gene expression studies, Northern blotting, hybridization, poly-A+ selection and much more.
Material not included
  • Chloroform p.A.
  • Isopropanol p.A.
  • 70 % Ethanol p.A.
  • Application Notes RNA Insta-Pure is a single-step method for the isolation of total RNA from tissues, cells, bacteria, plants and yeasts. The entire procedure takes 1®0150,2 hours and yields undergraded RNA that is free of protein and DNA contaminations. This single reagent allows very high RNA purification yield and purity, even for tissues and cells containing high amounts of RNases. The purified RNA contains the entire spectrum of RNA strands, including small (4®0150,5 S) RNAs. The ease of the RNA Insta-Pure method combined with excellent recovery from small quantities of tissues or cells makes this product especially suitable for gene expression studies, Northern blotting, hybridization, poly-A+ selection and much more.
    Comment

    Expected yields of RNA per mg of tissue (106 cells)

  • Liver and Spleen : 6-10 μg
  • Kidney : 3-4 μg
  • Skeletal muscles and brain : 1-1,5 μg
  • Placenta : 1-4 μg
  • Epithelial cells 8-15 μg
  • Fibroblasts : 5-7 μg
  • Assay Procedure

    RNA isolation by the RNA InstaPureTM method includes the following steps:

    1. Homogenization RNA InstaPureTM (2 ml/100 mg tissue or 107 cells)
    2. RNA Extraction 1 vol. homogenate + 0.1 vol. Chloroform
    3. RNA Precipitation 1 vol. isopropanol
    4. RNA Wash 70 % ethanol

    Homogenization:
    1. Homogenize tissue samples with RNA InstaPureTM (2 mL per 100 mg tissue) with a few strokes in a glass-Teflon homogenizer.
    2. To isolate RNA from cells grown in suspension, sediment cells and lyse them by the addition of 0.2 mL RNA InstaPureTM per 106 cells. Cells grown in monolayer are lysed directly in the culture dish by the addition of RNA InstaPureTM (1 mL per 3.5 cm petri dish). Solubilize RNA by passing the lysate a few times through the pipette.
    RNA Extraction:
    Add 0.2 mL chloroform per 2 mL of homogenate, cover tightly the samples, shake vigorously for 15 seconds and let them stay on ice (or at 4° C) for 5 minutes. After addition of chloroform and centrifugation t for 15 minutes at 12.000 g (4° C) the homogenate forms two phases: the lower yellow phenol-chloroform phase and the colorless upper aqueous phase whereas DNA and proteins are in the interphase and organic phase. The volume of the aqueous phase is about 50 % of the initial volume of RNA InstaPureTM plus the volume of tissue used for homogenization.

    RNA Precipitation:
    Transfer the aqueous phase to a fresh tube, add an equal volume of isopropanol, mix and store the samples for 15 minutes at 4° C. Centrifuge samples for 15 minutes at 12.000 g (4 °C). RNA precipitate (often invisible before centrifugation) forms a white-yellow pellet at the bottom of the tube.

    RNA Wash:
    Remove the supernatant and wash the RNA pellet once with 70 % ethanol by vortexing and subsequent centrifugation for 8 minutes at 7.500 g (4 °C). Repeat the washing step if necessary (phenol smell !). Use at least 0.8 mL of ethanol per 50-100 μg RNA.
    At the end of the procedure briefly dry the pellet under vacuum for 10 minutes. It is important not to let the RNA pellet dry completely, as it will greatly decrease its solubility. Dissolve the RNA pellet in 0.5 % SDS or in 1 mM EDTA, pH 7 solution by vortexing or by passing it a few times through a pipette tip. An incubation for 10-15 minutes at 60 °C may be required to dissolve preparations of RNA. Diethylpyrocarbonate (DEPC)- treated RNAse free solutions (3) should be used for RNA solubilization.

    Notes and Comments:
    • Isolation of RNA from a small amount of tissue (1-10 mg). Homogenize samples in 0.8 mL RNA InstaPureTM, transfer the homogenates to Eppendorf tubes, add 80 μL of chloroform and store samples for 5 minutes at 4 °C. Centrifuge samples in an Eppendorf centrifuge for 15 minutes, collect the aqueous phase and precipitate RNA with 0.4 mL of isopropanol for 45 minutes or overnight at 4 °C. Centrifuge RNA precipitates for 15 minutes and wash once with 0.8 mL of 70 % ethanol.
    • Following isopropanol addition store samples overnight at 4 °C in case the procedure has to be interrupted at this step.
    • An additional precipitation might be necessary to use RNA isolated by the RNA InstaPureTM method in enzymatic assays. Following solubilization precipitate RNA in the presence of 0.2 M NaCl with one volume of isopropanol or with two volumes of ethanol for 1 hour at -20 °C.
    • Hands and dust may be the major source of RNAse contamination. Use gloves and keep tubes closed. The use of sterile disposable polypropylene tubes is recommended throughout the procedure.
    • Some commercial SDS preparations have acid pH . Adjust SDS preparation to pH 6.5-7.5 if necessary.

    Restrictions For Research Use only
    Storage 4 °C
    Storage Comment 4°C. Do not freeze.
    Stable for nine month, refer to expiration date.
    Expiry Date 4-8 months