biotechnology northern blotting

7/28/2019 Biotechnology Northern Blotting

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Steps > RNA Isolation, Probe Generation > Electrophoresis, Transfer > Hybridization,

Detection

Northern analysis remains a standard method for detection andquantitation of mRNA levels despite the advent of powerful techniques,

such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message

abundance between samples on a single membrane. It is the preferredmethod for determining transcript size and for detecting alternatively

spliced transcripts.

The Northern blotting procedure is straightforward and providesopportunities to evaluate progress at various points (e.g., integrity of the

RNA sample and how efficiently it has transferred to the membrane). RNAsamples are first separated by size via electrophoresis in an agarose gel

under denaturing conditions. The RNA is then transferred to a membrane,

crosslinked and hybridized with a labeled probe. Northern hybridization isexceptionally versatile in that radiolabeled or nonisotopically labeled DNA,in vitro transcribed RNA and oligonucleotides can all be used as

hybridization probes. Additionally, sequences with only partial homology(e.g., cDNA from a diff erent species or genomic DNA fragments that might

contain an intron) may be used as probes.

Despite these advantages, there are limitations associated with Northernanalysis. First, if RNA samples are even slightly degraded, the quality of

the data and the ability to quantitate expression are severelycompromised. For example, even a single cleavage in 20% of 4 kb target

molecules will decrease the returned signal by 20%. Thus, RNase-freereagents and techniques are essential. Second, a standard Northern

procedure is, in general, less sensitive than nuclease protection assays and

RT-PCR, although improvements in sensitivity can be achieved by usinghigh specific activity antisense RNA probes, optimized hybridization buffersand positively charged nylon membranes. Sensitivity can be furtherimproved with oligo dT selection for enrichment of mRNA, since physical

constraints of gel electrophoresis and membrane transfer limit the amountof RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination

with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of

Northern blotting has been the difficulty associated with multiple probeanalysis. To detect more than one message, it is usually necessary to strip

the initial probe before hybridizing with a second probe. This process canbe time consuming and problematic, since harsh treatment is required to

strip conventional probes from blots.

Steps Involved in Northern Analysis

http://www.ambion.com/techlib/basics/northerns/index.html#2






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Steps Involved in Northern AnalysisSubmitted by:-Rashid hussain

Submitted to:-Dr.Siddharath Jetlie

Program:-B.Tech[Bio-technology]
























































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Steps Involved in Northern AnalysisRoll number:- A55,Reg no:-

10908206























































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Gratitude cannot be seen or expressed. It

can only be felt in heart and is beyond

























































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Steps Involved in Northern Analysisdescription. Often words are inadequate to

serve as a model of expression of one’s

feeling, specially the sense of indebtedness

and gratitude to all those who help us in

our duty.

























































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It is of immense pleasure and

profound privilege to express my gratitude

and indebtedness along with sincere thanks

to sir Dr. siddharath,lecturer of

























































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Steps Involved in Northern AnalysisBiotechnology at Lovely Professional

University for providing me the opportunity

to work for a project on “NORTHERN

BLOTTING” I am beholden to my family and
























































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Steps Involved in Northern Analysisfriends for their blessings and

encouragement.























































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1.HISTORY OF NORTHERN BLOTTING
























































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Steps Involved in Northern Analysis2.BACKGROUND INFORMATION-NORTHERN BLOT TECHNIQUE

3.NORTHERN BLOT METHOD

4.APPLICATION OF THE NORTHERN BLOT

5.DISADVANTAGE OF NORTHERN BLOT

























































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Steps Involved in Northern Analysis6.ADVANTAGE OF NORTHERN BLOT

7.NORTHERN BLOT PROTOCOL

8.MATERIAL NEEDED FOR NORTHERN BLOT PROTOCOL

9.RNA GEL

























































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Steps Involved in Northern Analysis10.GROSS LINKING NYLON MEMBRANE NORTHERN BLOT

11.PRE HYBRIDIZATION FOR NORTHERN BLOT

12.LABELING PROBE FOR NORTHERN BLOTTING

13.TIPS FOR NORTHERN BLOT

























































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Steps Involved in Northern Analysis14.NORTHERN BLOT REFRENCE























































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Steps Involved in Northern AnalysisHistory of NorthernBlotting

Northernblotting is an RNAblotting techniquewhich was

developedin 1977 by Alwineet al. at StanfordUniversity It was

namedaftertheSouthernblottechniquewhich blots for DNA,

inventedbyEdwin M. Southernin 1975.Westernblot, a methodfor


































http://www.molecularstation.com/dna/southern-blot/


http://www.molecularstation.com/protein/western-blot/

http://www.molecularstation.com/protein/western-blot/



















http://www.molecularstation.com/dna/southern-blot/





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Steps Involved in Northern Analysisblotting for proteinsis also a play on the Southernblot namingand

was namedin 1981

BackgroundInformation- NorthernBlot

Technique

























































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Steps Involved in Northern AnalysisNorthernanalysis despite its age in the high tech world of Real Time

PCR, nucleaseprotectionassays(RPAs) and microarrays, is still

the gold-standardfor the detectionand quantitationof mRNAlevels.

This is becausenorthernblot analysis allowsa direct comparisonof
























































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Steps Involved in Northern Analysisthe messengerRNAabundancebetweensampleson a single

membrane.

In northernblot the main differencebetweenthe other blotting

techniquesis that RNAis the factor being detected. Also, due to the
























































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Steps Involved in Northern Analysisfact that RNAis usually single-stranded,it createscomplex

secondarystructureswhich affect its migrationand hence

denaturingconditionsare used to run the gels (unlike Southern).























































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Steps Involved in Northern AnalysisRNAis separatedout by RNAgel electrophoresis(usually agarose

gel electrophoresis), subsequenttransfer to membrane,

hybridizationwith probe, and finally detection.

































http://www.molecularstation.com/rna/rna-gels/


























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Similarly to

Southern

blotting, the























































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Steps Involved in Northern Analysishybridization probes may be DNA or RNA in northern

blotting.

A variant of the procedure known as the reverse northern

blot was occasionally (although, infrequently) used. In this

procedure, the substrate nucleic acid (that is affixed to the

























































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Steps Involved in Northern Analysismembrane) is a collection of isolated DNA fragments, and

the probe is RNA extracted from a tissue and radioactively

labelled.

The use of DNA microarrays that have come into

widespread use in the late 1990s and early 2000s is more

























































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Steps Involved in Northern Analysisakin to the reverse procedure, in that they involve the use of

isolated DNA fragments affixed to a substrate, and

hybridization with a probe made from cellular RNA. Thus

the reverse procedure, though originally uncommon,

enabled the one-at-a-time study of gene expression using

























































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Steps Involved in Northern Analysisnorthern analysis to evolve into gene expression profiling,in

which many (possibly all) of the genes in an organism may

have their expression monitored























































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Northernblot method























































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Applicationsof the NorthernBlot
























































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Northern blots have been superceded in most areas by

Real Time PCR and microarray approaches. It is not often

used for clinical or diagnostic purposes.
























































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Steps Involved in Northern AnalysisThe northern blot protocol and its variations are used

however in molecular biology research to:

• a gold-standard for the direct study of gene expression at

the level of mRNA (messenger RNA transcripts).

• detection of mRNA transcript size


























































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Steps Involved in Northern Analysis• study RNA degradation

• study RNA splicing - can detect alternatively spliced

transcripts

• study RNA half-life

























































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Steps Involved in Northern Analysis• study IRES (internal ribosomal entry site) - to remove

possibility of RNA digestion vs 2nd cistron translation.

• often used to confirm and check transgenic / knockout

mice (animals)
























































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Steps Involved in Northern AnalysisDisadvantagesof NorthernBlotting

Often radioactivity is used. This preventsease of performing

it, use and disposal. New methodsof non-radioactivedetection
























































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Steps Involved in Northern Analysishave been generatedallowingnon-radioactivedetection. See

Pierce.

• The whole processof northernblotting takes a long time

usually, fromsamplepreparationthroughto detection.


























































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Steps Involved in Northern Analysis• If RNAsamplesare even slightly degradedby RNases, the

quality of the data and quantitationof expressionis quite

negativelyaffected.

• The standardnorthernblot methodis relatively less sensitive

than nucleaseprotectionassaysand RT-PCR. The sensitivity


























































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Steps Involved in Northern Analysisof northernblots may be increasedwith the use of nylon

positively-chargedmembranes,use of a highly specific

antisenseprobe.

• Detectionwith multiple probesis a problem.Often, the

membranesmust be strippedbefore hybridizationand


























































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Steps Involved in Northern Analysisdetectionwith a secondprobe. This is a problemas harsh

conditionsare requiredto strip off probesfrom the blot and is

also time consuming.Also, there is a limit to the amountof

timesa blot may be stripped.
























































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Steps Involved in Northern AnalysisAdvantagesof NorthernBlotting

The advantagesof northernblots include:

• It is awidely acceptedand well regardedmethod

• northernblotting is a straight-forwardmethod



























































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Steps Involved in Northern Analysis• Often it is used as a confirmationor check

• Often a gold-standard

• it is a versatile protocol as it can allow the usageof many

typesof probes(vs Real time PCR) including:radiolabeled


























































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Steps Involved in Northern Analysisand non-radiolabeled, in vitro transcribedRNAand even

oligonucleotidessuch as primers.

• Sequenceswith even partial homology,unlike real time PCR

or other methodscan be used as hybridizationprobes(i.e
























































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Steps Involved in Northern Analysissequencefrom different speciesfor homologyanalysis, or

even genomicfragmentscan be used).























































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Steps Involved in Northern AnalysisNorthernBlot Protocol

As mentionedthe steps in northernblotting include:

1. RNAisolation

2. Gel electrophoresisof RNAfor separation

























































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Steps Involved in Northern Analysis3. Transfer to membrane(usually positively chargednylon as RNA

is negativelycharged)

4. Cross-linking of RNAto membrane(usually by UV-crosslinking

or chemical means)
























































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Steps Involved in Northern Analysis5. Hybridization

6. Detection

Materials Neededfor NorthernBlot Protocol
























































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Steps Involved in Northern AnalysisBuffer Recipes

20XSSPE(500 ML)

• 3.6M NaCl : 105.2 g

• 0. 2M phosphatebuffer pH 7.0: 100mLof 1M



























































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Steps Involved in Northern Analysis• 20mMEDTA:20mLof 0.5M

PHOSPHATEBUFFERPH7.0 (500ML)

• NaH2PO4(monobasic)140 mL of 1M

• Na2H2PO4(Dibasic) 360 mL of 1M



























































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Steps Involved in Northern Analysis• pH to 7.0 after both are added

HYBRIDIZATIONBUFFER=HB (100MLS)

• 5X SSPE: 25mls 20X

• 2% SDS: 20 mls 10%



























































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* *Right before using add 1000ugdenaturedRNAsefree CT DNA

(heat to 95o for 3 min to denature) 5000ugyeast RNA
























































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Steps Involved in Northern AnalysisWASH:2X SSPE+ 0.1% SDS (500 mls) 50 mls 20X 5 mls 10% SDS=

0.1%SDS

RUNNINGBUFFERFOR RNAGEL (1L)

• 100 mls 10X MOPS


























































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Steps Involved in Northern Analysis• 52. 6 mls of formaldehyde

• 847. 4 mls H20

RNAGEL (70ML)

• 0.84 gagarose


























































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Steps Involved in Northern Analysis• 7 mls 10x MOPS

• 58.38 mls H2O

• Put gel in solutionby heating. Let gel cool to 60°C, then add

Formaldehydeto equal 0.66M--3.78 mls


























































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Steps Involved in Northern Analysis• Pour gel in fumehood if possible

RNA Gel
























































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Steps Involved in Northern AnalysisAfter isolatingRNA, the RNAmust be separatedout on a gel

(usually agarose).

NorthernBlot Transfer to Nylon MembraneProtocol

After runningthe RNAgel, washthe gel with distilledwater put on posiblotter.




























































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Steps Involved in Northern AnalysisLayersfromtop to bottomare:

• sponge

• 3-4 Whatmanpaperscut to size ( both of these up aboveshouldbe soakedin 10X

SSPEbut not dripping)

• gel mask.


























































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Steps Involved in Northern Analysis• Note: Makesure the gel overlaysthe maskso that it can seal membranewith line of

the top of gel and the right top corner marked3 piecesslightly bigger3M paper

hard plastic with holes

Clampon and makesure there is a goodseal. Use 75-80 mmHg of pressurefor 1 hr or

more.

























































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Steps Involved in Northern AnalysisBlot membranedry

Cross-linking Nylon Membranes- NorthernBlot Protocol

• Cut top right corner wrap in saran wrap.

• UV irradiate with RNAside downfor 2.5 min.



























































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Steps Involved in Northern Analysis• Washfor a couple of minutesin hot 2X SSPE/0.1%SDS

solution(Microwavesolutionfor 30-45 secondsat 50% power.

• Air dry























































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Pre Hybridizationfor NorthernBlot

1. Pre Hybridizefor 6 hours-overnight

2. Set oven to 65°C heat HB buffer to put SDSin solution
























































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Steps Involved in Northern Analysis3. Roll up membraneinside piece of meshand put into hybridizaton

tube(2XSSPE/0.1%SDS)

4. Checkfor air bubbles

5. Put tube in oven balancedwith anothertube on oppositeside
























































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Steps Involved in Northern AnalysisLabelingprobefor NorthernBlotting

1. Put 25ng of probein a total volumeof 11uL with ddH2O

2. Denature@ 95°C 3 min. This is to get the probesingle strandedand

removesecondarystructurewhichwouldpreventefficient

hybridization.

























































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Steps Involved in Northern Analysis3. Quick cool on wet ice. This is to keep the probesingle stranded,free of

secondarystructureand not allowreannealing(or reformingof

secondarystructures).

4. Add 4ul High Prime(BoehringerMannheim)5ul alpha radiolabeledP-32

dCTP3000uCi/mmole20ul total

























































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Steps Involved in Northern Analysis5. Incubate10-15 min 37°C

6. Add 28uL of 1X TE, 2ul 0.5M EDTA,to 20ul reaction.

7. Pre-spin G-25 Sephadexcolumnfor 1 minute.























































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Steps Involved in Northern Analysis8. Add 50ul sampleto columnand spin for 2.5 min in clinical centrifuge.

This is to purify probeaway fromlabel.

9. Throwaway column.

10.Mix in anew tube 100ugCT-DNA50ug Yeast RNAin a 0.5mLtube.
























































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Steps Involved in Northern Analysis11.Denature95°3 min

12.Add to hybrid tube mix, hybridizeat 65°C for 20-36 hrs

Post hybridizationProtocolfor NorthernBlotting
























































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Steps Involved in Northern AnalysisHybridizefor 36-48 hours then wash.

1. Heat up 2X SSPE/0.1%SDS (wash) heat up to 65°C (Use microwaveor

water bath to warmsolution)

2. Take out tube and pour liquid into hot radioactivewaste container.

3. Rinse with 10ml washdo this twice and pour waste out.

























































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Steps Involved in Northern Analysis4. Put in 40 to 50 mls of washand shakevigoursly.

5. Put in hybridizationoven for 20 min rotating.

6. Pour downsink

7. Another40-50mls and shakein oven.
























































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Steps Involved in Northern Analysis8. Pour out to waste container(sink if not hot or toxic) and makesure it is

no longer hot and then take out membrane.

9. Washon shaker with 0.2XSSPEw/ 0.1%SDS at RT for 15 min.

10.Air dry
























































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Steps Involved in Northern Analysis11.Expose

Tips for NorthernBlot

If you currently have a preferredmethodfor isolatingRNA, continueto use it.

























































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Steps Involved in Northern AnalysisAlwaysrun an agarosegel of your RNAto assessthe quality. Do not only rely

on spectrophotometryresults.

* Use DEPCtreatedwater and bakedglassware.This solution, absolutely,

positively must be Rnasefree. Rnaseis everywhere!Wear gloves, use only
























































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Steps Involved in Northern Analysisbakedglassware,or virgin plastic. DePCtreat your water, buffers (except

Tris). Be very careful..

NorthernBlot References
























































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Steps Involved in Northern Analysis1. AlwineJC, KempDJ, Stark GR (1977). "Methodfor detectionof specific RNAsin

agarosegels by transfer to diazobenzyloxymethyl-paper and hybridizationwith

DNAprobes". Proc. Natl. Acad. Sci. U.S.A. 74 (12): 5350-4.

2. W. Neal Burnette(April 1981). "'Westernblotting': electrophoretictransferof

proteinsfromsodiumdodecyl sulfate — polyacrylamidegels to unmodified

nitrocelluloseand radiographicdetectionwith antibodyand radioiodinatedprotein

A". AnalBiochem112 (2): 195-203.


























































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Steps Involved in Northern Analysis3. http://www.molecularstation.com/rna/northern-blot/

4. http://en.wikipedia.org/wiki/Northern_blot

5. http://www.ambion.com/techlib/basics/northerns/index.html

































http://www.molecularstation.com/rna/northern-blot/


http://en.wikipedia.org/wiki/Northern_blot

http://www.ambion.com/techlib/basics/northerns/index.html



http://en.wikipedia.org/wiki/Northern_blot
























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