Molecular Techniques and Methods

Northern Analysis of Total RNA and mRNA

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION
This protocol is for hybridization between 100% matched sequences.
For hybridization between heterologous sequences (less than 90% matches), use low stringent condition.


MATERIALS AND SOLUTIONS
10 x MOPS Buffer (500 ml)
200 mM MOPSpH 7.0 --------------------------------- 21 g
80 mM Sodium acetate -------------------------------- 13.5 ml of 3 M Sodium acetate
10 mM EDTA ----------------------------------------- 10 ml of 0.5 M EDTA
Add DEPC-trated H2O to make a final volume of --- 500 ml


Electrophoresis buffer (1 liter)
10 X MOPS buffer ------------------------------------ 100 ml
37% Formaldehyde ------------------------------------ 82 ml
Distilled H2O ------------------------------------------- 818 ml
  • Formaldehyde interacts with O2 to become formic acid. Minimize air-exposure.


    2x Gel Loading Buffer (1 ml)
    50% Deionized Formamide ----------------------------- 500 ul of 100% Formamide
    12% Formaldehyde ------------------------------------ 334 ul of 37% Formaldehyde
    0.1% Xylene cyanol ---------------------------------- 20 ul of 5% Xylene cyanol
    0.1% Bromophenol blue ------------------------------- 20 ul of 5% Bromophenol blue
    1 mM EDTA ------------------------------------------- 2 ul of 0.5 M EDTA
    0.025% SDS ------------------------------------------ 2.5 ul of 10% SDS
    12.5% Glycerol -------------------------------------- 125 ul of 100% Glycerol


    Ethidium bromide Staining Solution (100 ml)
    100 ug Ethidium bromide ------------------------------- 10 ul of Ethidium bromide Stock Solution (10 mg/ml)
    100 mM Ammonium acetate ------------------------- 1 ml of 10 M Ammonium acetate
    DEPC-trated H2O ------------------------------------- 99 ml
  • Desolve until background fluorescence is minimal.
  • Keep in aluminum foil-covered bottle.


    Prehybridization Solution (20 ml)
    50% Deionized Formamide ----------------------- 10 ml of Deionized Formamide
    25 mM PIPESpH 6.8 ----------------------------- 0.5 ml of 1 M PIPES
    5 mM EDTA ---------------------------------------- 0.2 ml of 0.5 M EDTA
    10 X Denhardt's solution -------------------------- 2 ml of 100 X Denhardt's solution
    0.75 M NaCl ---------------------------------------- 3 ml of 5 M NaCl
    1% SDS ------------------------------------------- 2 ml 10% SDS
    2 mg Salmon Sperm DNA ------------------------ 0.2 ml of Salmon Sperm DNA (10 mg/ml)
    DEPC-trated H2O -------------------------------- 2 ml




    PROCEDURES

    Gel Preparation

    1. Prepare formaldehyde agarose gel as follow.

    Target mRNA > 2kb
    0.8%
    Agarose
    0.48 g
    DEPC-H2O
    44 ml
    10x MOPS Buffer
    6 ml


    Target mRNA < 2kb
    1%
    Agarose
    0.6 g
    DEPC-H2O
    44 ml
    10x MOPS Buffer
    6 ml

    2. Cool the agarose to 65oC.

    3. In a fume hood, add 10 ml of a 37% Formaldehyde solution just prior to pouring the gel.
  • Formaldehyde is TOXIC. Work in a fume hood!


    Denaturation of RNA

    4. Mix 10-20 ug total RNA (or, 1-2 ug poly A+-RNA) with equal volume of 2x Gel Loadling BUffer.

    5. Denature RNA for 5 minutes at 90oC.

    6. Cool down RNA on ice.
  • This step prevents the secondary structure formation.


    Electrophoresis and Blotting

    7. Load the RNA sample in the gel and electrophorese the RNA in Electrophoresis buffer at 4-5 V/cm until the BPB migrated 8 cm (4-5 hours) in a fume hood.
  • Optional: After electrophoresis, stain the gel for 10 minutes in Ethidium bromide Staining Solution. Photograph the gel.
  • Optional: If the RNA to be analyzed is >2.5kb in length, treat the gel with alkaline solution to partially hydrolyze RNA.
  • Caution, too much treatment can degrade RNA completely.
    (a) Soak the gel for 5 min in 300 ml (5 gel vol) of 0.05N NaOH.
    (b) Soak the gel for 30 min in 600 ml (10 gel vol) of 20x SSC.

    8. Transfer the RNA to uncharged, neutral nylon membrane for 1 hr.
  • Use neutral, uncharged nylon membrane.
  • Positively charged nylon membrane will give high background and also difficult to get signal.

    9. Remove nylon membrane from the gel.

    10. Fix the RNA on nylone membrane by UV light (or, in the 80oC oven for 2 hours).


    Hybridization Conditions

    11. Prehybridize RNA blotted-nylon membrane for 2 hours at 35-42oC in 10 ml of Prehybridization Solution.

    12. Replace with a new Prehybridization Solution (50-100 ul/ cm2 of membrane) with radioactive-labeled probe.
  • For detection of low abundant message, add at least 0.1 ug (2x 107cpm) denatured, single strand probe.
  • If probe is ds-DNA, it should be denatured prior to add hybridization solution.
  • Add 100 ul H2O and 100 ul Salmon Sperm DNA to probe and heat denature at 95oC for 5 min.
  • Add denatured probe immediately for hybridization.

    13. Hybridize overnight at 35-42oC.

    14. Wash membrane in two changes of 2x SSC, 0.1% SDS at room temperature and two changes of 0.1 x SSC, 0.1% SDS at 65oC

    15. Perform autoradiography by exposing the X-ray film on the membrane.
  • (If nonradioactive-labeled probe used, follow the chemiluminescent detection protocol.)



  • NOTES

  • For heterologous sequence hybridization, use low stringent condition.
    Ex) Increase NaCl concentration to 1 M in prehybridization solution.
    Decrease hybrization temperature to 30-35oC.
    Wash membrane at room temperature.


    KIT INFORMATION



    REFERENCES


  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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