Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting...

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Laboratory Techniques I Oncology for Scientists I September 8 th , 2016 Hayley Affronti, PhD Student [email protected] Dr. Sheila Figel too!

Transcript of Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting...

Page 1: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

LaboratoryTechniquesIOncologyforScientistsI

September8th,2016HayleyAffronti,PhDStudent

[email protected]

Dr.SheilaFigeltoo!

mailto:[email protected]
Page 2: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

“Whenwefirsthitthelabtherearesomanythingstolearnbeforeweevengetstartedthatmanythingsgounlearned”–BiteSizeBio

Page 3: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

Overview

• ExperimentalPrinciples• Molecularbiologytechniques,thateveryoneneeds!– DNA-PCR– RNA-rt-PCR– Protein-westernblotting

• Cellculture

Page 4: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

ExperimentalPrinciples

• Controls– Negative– Positive

• Quantification– QualitativevsQuantitativedata

• Technicalreplicatesvs.biologicalreplicates

Page 5: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

Qualitativevs.Quantitative• Qualitative

– “Quality”– Achangeinappearanceor

othercharacteristichasbeenobserved

– Descriptive– “Cellshavebecomeelongated

andfibroblastic”

• Quantitative– “quantity”– Achangeinsomeparameter

thatyouhavemeasured– Objectivelymeasured– “92%ofthecellsshowan

elongatedphenotypeasindicatedbyalength-to-widthratioofgreaterthan4.”

www.proteomesci.com

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Replicates

• Technicalreplicates– Withinasingleexperiment– Makemeasurementsfromthesamesourceatthesametime

– Demonstratesconsistencyintechnique• Biologicalreplicates– Multiplerepetitionsofthesameexperiment– Demonstratesconsistencyinexperimentalresults

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Replicates

Example:wound-healingassay

• Technicalreplicates– Threeindependent

measurementsalongthelengthofthe“wound”

• Biologicalreplicates– Repeattheexperimentthree

times

1

2

3

0

2.5

5

7.5

10

1 2

Page 8: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

Basicmoleculartechniquesthateveryoneneeds!!!

And….Moreimportantly

Page 9: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

DNA,RNAandprotein

• DNA– Southernblot– PCR

• RNA– Northernblot– RT-PCR,qPCR

• Protein– Westernblot

DNAà RNAà Protein

Page 10: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

DNA

• Oldertechniquesallowyes/nodetectionofgenecopiesorchromosomalrearrangements– Fluorescenceinsituhybrization(FISH)– Spectralkaryotyping(SKY)– Southernblotting

• Moderntechniquesallowsequenceanalysis– PCR– Genomesequencing

SKY

FISH

Page 11: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

ExtractionofgenomicDNA

1. CellLysis!– Highconcentrationsofchaotropicsalt(HCl,guanidinethiocyanate,

urea,andlithiumperchlorate)– Detergents– Enzymes(ProteinaseK)

2. DNApurification– Phenol/Chloroformextraction

• Denaturedproteinsinthephenollayer• EthanolprecipitationofDNAinaqueouslayer

Page 12: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

Howdoesitwork!?

Page 13: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

ExtractionofPlasmidDNA

• PlasmidsmaintainedinE.coli• Alkalinelysis

– Step1–resuspendbacteria– Step2–lysis/denaturation

• NaOH/SDS–burstscells&denaturesDNA(bacterialchr.&plasmid)

– Step3–precipitationofprotein/bactDNA• Potassiumacetate–plasmidDNA

renatures– Step4–ethanolprecipitationof

plasmidDNA

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PCR

• AmplificationsofsmallsegmentsofDNA• Usedtoidentifymutations,cloning,expression(qPCR)

Page 15: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

MaterialsforPCR

• PCRreactionmixture:– DNAtemplate

• gDNA• PlasmidDNA• cDNA(RT-PCR)

– Primers– Polymerase

• Taq

– Buffer– Thermalcycler

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StepsforPCR

• Denaturation– SeparatethestrandsofDNA– Heatto95or98°

• Annealing– PrimerbindingtoDNA– Temperaturevaries– DependsontheTmofprimer– Typically5°lessthanthelowestTmoftheprimer

• Elongation– PolymerasebindstoprimedDNAandaddsnucleotides

• Cycles– Typically~30

Page 17: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

AgaroseGelElectrophoresis

• Electrophoresis– Migrationofmoleculesthroughamatrixbasedonsize&charge

– Matrixissolidbutporous• Agarose– DNA,RNA– Restrictiondigest

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TypesorPCR1. StandardPCR– AmplifyDNA(genomicorplasmid)

2. ReversetranscriptionPCR(RT-PCR)– AmplifycDNA

3. qPCR– QuantitativePCR– AmplifyDNAorRNA

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rt-PCR• RNAextraction(Trizol)

• ReversetranscriptionPCR

• Semi-quantitative–comparesignalsongel

• DetectcDNAs(indicativeofRNA)

• Steps:– Reversetranscription

• RNAà cDNA– PCRusingcDNAtemplate

Naitoetal,2004

Page 20: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

qPCR(Real-TimePCR)• QuanititativePCR• Amplifyshortsegmentrepresentinga

gene• DNA-bindingdye

– SYBRGreen• Excitation=488nm• Emission=522nm

• FluorescenceofdsDNAmeasuredaftereachcycle

• Relativequantification– Compareamplifiedamttothatof

housekeepinggeneCt=Thresholdcycle

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QuantitativePCR• ApplyqPCRtocDNAstoquantifyRNAlevels• ThisreplacesolderRNAdetectiontechniquessuchasnorthernblotting(whichissemi-quantitative)

Page 22: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

PCRexperimentalprinciples• Controls

– Amplificationofhousekeepinggene(RT-PCR&qPCR)– Positive–DNAtemplateknowntocontainthecorrectsequence– Negative–Reactionmixture+water(notemplate)

• Quantification– StandardPCRisqualitative–presence/absenceofband,sequencedata– RT-PCRissemi-quantitative–bandintensitiescanbecomparedbasedon

equivalentcontrolsignalforeachsample– qPCRisquantitative–Ctvaluescanbecompared

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Westernblot• Allowscomparisonoflevelsofproteinin

samples• Ex:Isexpressionofacertainprotein

decreasedwhencellsaretreatedwithadrug?

• Steps– Isolationofprotein– RunlysatesthroughSDS-PAGEgel– Transferproteinfromgeltomembrane– Incubatemembranewith1°/2°

antibodies– Detectsignalviafilm Leeetal,2011

Page 24: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

WB-isolationofprotein• Cellsortissue• RIPA(radioimmunoprecipitationlysisassaybuffer)

– ContainsSDS&sodiumdeoxycholate–ionicdetergents– Disruptsmembranes&protein-proteininteractions– proteinaseinhibitors

• Quantificationoftotalproteininlysate– Bradfordassay

• Coomassiedyeturnsfromredtobluewhenbindingprotein• ReadA595• Comparetostandardcurvetodetermineprotein

concentration• ug/ul

Page 25: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

WB-SDSPAGE• SDS=sodiumdodecylsulfate

– Detergent• PAGE=polyacrylamidegelelectrophoresis

• PreparationofSDS-PAGEgel– Acrylamide:bisacrylamide(whatpolymerizes)

• 7.5%gel,10%gel,etc– SDS(denaturant)– Buffer(maintainspH)– APS/TEMED(initiatespolymerization)

Page 26: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

Theelements,becausethisisprettycool…

+

=

GlycineProteinsCl-

ProteinsGlycine

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WB-transfer

• Transferproteinfromgeltomembrane– PVDFornitrocellulose

• Hydrophobic– Assemble“sandwich”– Electroblotting

Page 28: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

WB–detectionvia1°/2°antibodies

• Blockmembrane– Preventnon-specificbindingofantibodies– BSA,non-fatmilk

• 1°antibody– Specificforproteinofinterest

• 2°antibody– Specificforthespeciesofthe1°Ab– ConjugatedtoafluorortoHRP(horseradishperoxidase)

Page 29: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

WB–detectionvia1°/2°antibodies• Fluor-conjugated2°Ab

– Scanmembrane&detectfluorescence• HRP-conjugated2°Ab

– IncubatemembranewithECLreagent– ExposemembranetoX-rayfilm

Leeetal,2011

Page 30: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

WBexperimentalprinciples• Controls

– Immunoblotting(samemembrane)forhousekeepinggene(ex:GAPDH)• Loadingcontrol

– Positive–Proteinlysatefromknownpositivesample– Negative–Dependentonexperiment

• Ex:TreatmentofcontrolcellswithPBSinsteadofdrug• Ex:Treatedcellsat“0h”(beforedrugcanhaveaneffect)

• Quantification– WBissemi-quantitative–bandintensitiescanbecomparedbasedon

equivalentcontrolsignalforeachsample– Inordertoquantify,applydensitometryanalysis

Page 31: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

Densitometry

• Allowsquantificationofimagesbasedonpixelintensity

• Awaytoquantifyrt-PCRandWB

UsingImageJ:• Measurepixelintensityofallbands• Normalizethevalueofeachbandto

thevalueofitsassociatedactinband• Comparenormalizedvalues

Caoetal,2011

Page 32: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

CellCulture• 1900s–tissueculture

– Harrison&Carrel– “amethodforstudyingthebehaviorofanimalcellsfreeofsystemic

variationsthatmightariseintheanimalbothduringnormalhomeostasisandunderthestressofanexperiment”

• 1952–developmentofcontinuoushumantumorcellline– HeLa

• Usesofculturedcells:– Developmentofantiviralvaccines– Productionofmonoclonalantibodies– Productionofcellproducts

• Insulin,HGH,interferon

– Understandingofneoplasia

Page 33: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

CellCulture• Benefits– Cancarefullycontrolenvironment– Preservation

– Avoidusinganimals– Rapid,relativelycheap

• Disadvantages/limitations– Expertise– Identificationofcelltype– Genetic&phenotypicinstability– Quantity

Page 34: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

CellCulture-TypesofCells

• Primaryculture– Cellsisolatedfromtumorsororgans– Non-immortalized– Replicationlimità senescence

• Immortalizedcelllines– Haveevadedsenescence– Sources

• Cancer–ex,HeLa,A549• Stableexpressionofagenewhichde-regulatescellcycle

– AdenovirusE1inHEK293– Telomerase

Primarycells–mouseembryonicfibroblasts

Immortalizedcells–HeLa

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CellCulture-Typesofcells

• Adherent– Epithelial&fibroblast

• Non-adherent– Hematopoieticcells– Jurkatcells–humanTlymphocytes

Page 36: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

CellCultureBasics• Growthmedia– DMEM,MEM,RPMI1640

• Aminoacids,vitamins,glucose– Phenolred–pHindicator– Serum–FBS,FCS,BS

• Proteins&polypeptides,growthfactors,aminoacids,lipids,carbohydrates,polyamines,urea,inorganics,hormones,vitamins

• Containsantitrypsinactivity

Thaietal,2014

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CellCultureBasics–adherentcells

• Plates/flasks– “Tissueculturetreated”

• Platesaremadeofpolystyrenewhichishydrophobic

• TCtreatment(manytypes)à makesurfacehydrophilic/negativelycharged

• Celladhesionproteins(ex,vitronectin&fibronectin)cancoatplate

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CellCulturebasics–adherentcells

• Subculturing– Trypsin/EDTA– Trypsin

• Serineprotease• Cleavesadhesionproteins(integrins)• Optimalactivityat37°

– EDTA• Chelatingagent–Calcium&Magnesium

– Neutralizewithcomplete(serum-containing)medium

Page 39: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

AsepticTechnique

• Cellculturemustbekeptsterile

• Freeofmicroorganisms– Bacteria– Fungi– Viruses

• Aseptictechnique–designedtocreateabarrierbetweenthesterilecellculture&microorganismsintheenvironment– Sterileworkarea– Goodpersonalhygiene– Sterilereagents&media– Sterilehandling

Page 40: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

SummaryI• Cell&moleculartechniquesallowscientiststoexaminesubcellularcomponents

• Eachexperimentshouldincludepositive&negativecontrols

• Measurementscanbequalitativeorquantitative• Technicalreplicatesensureconsistencywithinanexperiment;biologicalreplicatesprovideconfidenceinexperimentalresults

• DNAmaybeextractedusingphenol/chloroformor(forplasmids)alkalinelysis

Page 41: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

SummaryII• PCRallowsustoamplifypiecesofDNAorcDNA• Westernblottingallowsdetectionofproteinswithincellsortissue

• Densitometryenablesquantificationofotherwisesemi-quantitativedatathroughanalysisofimagepixelintensity

• Cellcultureisawaytostudycellsinthelaboratory• Aseptictechniqueiscriticalincellculture

Page 42: Laboratory Techniques I - Buffalo, NY · Laboratory Techniques I ... – Protein-western blotting • Cell culture. ... – Southern blot – PCR • RNA – Northern blot – RT-PCR,

Thankyou!Questions???


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