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  • Northern Blotting: Vuong Dang Thai Pham Truong Giang Tran Khanh Linh Dang Ngoc Linh

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    Northern blotting is a technique used to detect specific RNA sequences in a sample. It involves separating RNA fragments by size through gel electrophoresis, transferring them to a membrane, then using a radiolabeled probe to detect sequences that bind to it through hybridization. A case study demonstrated using northern blotting to compare expression levels of promoters in the mouse Igf2 gene. It has advantages of simplicity and specificity but is time-consuming and requires handling of radioactive materials.

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    Northern blotting is a technique used to detect specific RNA sequences in a sample. It involves separating RNA fragments by size through gel electrophoresis, transferring them to a membrane, then using a radiolabeled probe to detect sequences that bind to it through hybridization. A case study demonstrated using northern blotting to compare expression levels of promoters in the mouse Igf2 gene. It has advantages of simplicity and specificity but is time-consuming and requires handling of radioactive materials.

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    12 views18 pages

    Northern Blotting: Vuong Dang Thai Pham Truong Giang Tran Khanh Linh Dang Ngoc Linh

    Uploaded by

    My Hồ Thị Trà
    Northern blotting is a technique used to detect specific RNA sequences in a sample. It involves separating RNA fragments by size through gel electrophoresis, transferring them to a membrane, then using a radiolabeled probe to detect sequences that bind to it through hybridization. A case study demonstrated using northern blotting to compare expression levels of promoters in the mouse Igf2 gene. It has advantages of simplicity and specificity but is time-consuming and requires handling of radioactive materials.

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    of 18

    NORTHERN

    BLOTTING
    Vuong Dang Thai
    Pham Truong Giang
    Tran Khanh Linh
    Dang Ngoc Linh
    definition application

    case study
    principle
    OUTLINES
    procedure benefits/drawbacks
    DEFINITION
    Northern blot hybrid method is
    applicable to RNA, DNA-RNA or
    RNA-RNA hybrid. 

    Used to determine the size and


    content of a specific mRNA in
    an RNA mixture
    PRINCIPLE 05 Based on the principle of
    complementarity A-T, G-C.

    04 Identified gene expression

    03 Radiolabeled DNA PROBE hybrid with RNA destination

    02
    Prevents structure formation RNA's quadratic
    Denaturation Does not interfere with the transfer
    as well as the separation of RNAs on

    01
    the GEL
    RNA analysis by size -> electrophoresis
    Step of Northern Blotting
    Step1: RNA gel electrophoresis

    Step2: Blotting RNA

    Step3: Incubate membrane

    Step4: Autoradiography
    CASE STUDY: MOUSE
    Experimental problem and purpose:
    + About gene igf2
    + Gene is expressed by many promoters
    + Compare gene expression levels via promoter 

    Prepare:
    *Tissue samples were obtained from wild-type pups
    at 2.5 days of birth, treated with liquid nitrogen and
    preserved at -20C for RNA extraction
    Igf2 gene
    Step 1: RNA gel electrophoresis

    Isolated total RNA

    RNA (total RNA or mRNA only) separated by size by agarose gel electrophoresis
    Denaturation of RNA ribbons on gels by dd formaldehyde
    Step 2: Blotting (RNA)
    Step 2: Blotting (RNA)
    Step 3: Incubate membrane
    Step 3: Incubate membrane
    Step 4: Autoradiography
    CASE STUDY: MOUSE
    Result:

    + The extracted DNA/RNA has a high concentration, is clean, has few


    breaks and is concentrated in clear bands

    + Analysis of the Pm  promoter has 4 CpG islands and contains 159 nu. Bait
    specific for Pm was designed and amplified correctly in size from cDNA (159
    bp).

    + Pm has the expression level was lower than P3, P2 and higher than some
    other promoters of Igf2 gene.
    ADVANTAGES/ DISADVANTAGES:
    Advantages Disadvantages
    - The strength of this method is its - Time consuming procedure.
    simplicity. - RNA samples can be degraded by RNases.
    - Relatively high specificity - The chemicals used can be dangerous for
    the operator
    - The quality and quantity of RNA
    can be measured on the gel prior to
    blotting.
    - mRNA transcript size can be
    detected.
    - Use of radio active probes.

    APPLICATION
    - Detection of specific mRNA in a sample.
    - Used in screening of recombinants by detecting the mRNA produced by
    the transgene.
    - In disease diagnosis.
    - In gene expression studies.
    - RNA splicing studies.
    REFERENCES
    http://huib.hueuni.edu.vn/wp-content/uploads/2020/12/60-66.pdf?
    fbclid=IwAR3FgPZrZ85ZsK5YeZ34JSAuWHnheZI5eD1r6PtKcVAhizCQpJ1ilUeCHcQ

    https://luanvan.net.vn/luan-van/de-tai-phuong-phap-lai-northern-blot-70164/

    https://en.wikipedia.org/wiki/Northern_blot
    THANK YOU FOR
    YOUR TIME!

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