Northern Blotting

Content

Introduction

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Materials for northern blot

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Method for northern blot

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Prepare RNA Sample for Loading

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Visualise integrity of RNA by UV illiumination/EtBr staining 5
Transfer to nylon membrane

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Hybridization 7

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Northern Blotting INTRODUCTION Northern blot technique is very useful in the analysis of RNA molecules. 2 . it is stained with ethidium bromide and visualised using ultra violet light. the distance of migration is not linear. Northern blot analysis allows the detection and quantification of specific RNA species from a particular cell type. Isolated RNA is electrophoresed through an agarose/formaldehyde gel which separates the RNA species by size. the separate RNA in the gel is then transferred to a solid matrix such as filter paper through the blotting process. The probe is a DNA or RNA molecule which is chemically or radioactively labelled. it is first necessary to transfer the RNA from the agarose/formaldehyde gel to a nylon membrane. Generally this technique involves the isolation of the RNA molecules from sources of interest and the separation of the isolated RNA by agarose or polyacrylamide gel electrophoresis. RNA is detected by hybridisation using a labelled probe. When RNA has separated following electrophoresis. rather it is inversely proportional to the size RNA molecule. The faster migrating RNA fragments are the smallest. This analysis is the RNA blotting like southern blot. To probe for a specific mRNA species by northern blot. however.

10 mM EDTA)  Formaldehyde  Loading dye  Formamide  Ethidium bromide  0.0.2 M MOPS pH 7. 3M NaCl)  Saran wrap  Glass plate  Whatmann 3MM paper  Glass as weight for blot  Sponge soaked in 20 x SSC 3 .3M Na3citrate.5 M iodoacetamide  Microfuge  UV transilluminator  Hybond N (Nylon) membrane  20 x SSC (0.Northern Blotting MATERIALS FOR NORTHERN BLOT  RNase free water  Agarose  10X running buffer (0.

0 e. Prepare RNA Sample for Loading a. Pipetting the solution 2. d.Northern Blotting METHOD FOR NORTHERN BLOT 1. Preventing RNA degradation by high temperatures c. Adding formaldehyde (2. Adding loading dye f. Adding MOPS  MOPS is the most commonly used buffer for RNA gels due to its high buffering capacity at pH 7. Add 10-20 µg of RNA to a sterile eppendorf tube b. Load samples into the wells and run at 100 volts for 2 hours  The RNA samples are most commonly separated on agarose gels containing formaldehyde as a denaturing agent for the RNA to limit secondary structure 4 .2 M)  Formaldehyde is used for denaturing agent for the RNA to limit secondary structure. Adding formamide   Formamide lowers annealing temperature of the probe-RNA interaction.

Set up transfer b. Add transfer buffer d. Place the gel 5 . 4. Soak the paper e. Transfer to nylon membrane  A nylon membrane with a positive charge is the most effective for use in northern blotting since the negatively charged nucleic acids have a high affinity for them a. Place filter paper f.Northern Blotting 3. Place whatman 3 mm filter paper c. Visualise integrity of RNA by UV illiumination/EtBr staining  The gels can be stained with ethidium bromide (EtBr) and viewed under UV light to observe the quality and quantity of RNA before blotting. Remove any air bubbles g.

Flood the membrane with transfer buffer j. Put a nylon membrane on the gel i. Stack paper towels above the gel n.Northern Blotting h. Cover with whatman 3 mm filter paper l. Disassemble the transfer system 6 . Cover with plastic wrap m. Remove any air bubbles k. Allow transfer to go overnight 5.

Prehybridize membrane in prehybridize buffer b. Hybridization a. Discard the prehybridizied buffer d. Incubate for 2-4 hours c. Fix the RNA to nylon membranes using UV crosslinker 6. Addition of probe 7 . Remove paper towel and filter papers b. Remove paper towel and filter papers c.Northern Blotting a. Add hybridisation buffer 7.

or oligonucleotides with a minimum of 25 complementary bases to the target sequence. Membrane is ready to expose to film 8 . Incubate at 52 C for 30 minutes in hybridisation chamber (repeat 3 times) e. Add wash solution d. RNA.Northern Blotting  Probes for northern blotting are composed of nucleic acids with a complementary sequence to all or part of the RNA of interest. Remove the membrane 8. Incubate overnight in hybridisation chamber b. they can be DNA. discard the solution c. After incubation. a.

Northern Blotting 9 .