module 2: measuring gene expressionmeasurebiology.org/w/images/8/8a/sp19_m2d5_lecture_slides.pdf ·...
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Module2:Measuringgeneexpression
DecipheringtheetoposideresponseinDLD-1andBRCA2-/-cells
03/21/19
HRandNHEJrepairdouble-strandbreaks
ParadoxicalrelaFonshipexistsbetweencanceroccurrenceandtreatment
NatRev(2012)9:144-155
HR NHEJ
IfcancercellscarryamutaFonin
BRCA2,thencellsaredependentonNHEJfordouble-strandbreakrepair
IftreatmenttargetsDNAPKs,thencancercellsareunabletorepairdouble-strand
breaks
Workflowforcellviabilityexperiment
seedcells:DLD-1=25K
BRCA2-/-=50K
treatwithetoposide,100μM
incubate,24hr
incubate,1hr
replacemedia
incubate,48hr
Etoposidetreatmentyieldedunexpectedresults!
Your1staimistouseRNA-seqdatatoidenFfyeffect(s)of:• BRCA2-/-mutaFon• etoposidetreatment
Literatureconflictswithourdata
• IncreasingconcentraFonsofetoposideshowntopreferenFallykillBRCA2nullcancercells
• Previousclassused100μM(100μmol/L)!
Hucl,T.etal.(2008)CancerRes68:5023-30
Your2ndaimistodesignanexperimentthatwilladdresstheSp18results
• Whatexperimentalparametersmayexplaintheresults?
• Whatbiologicalresponsesmayexplaintheresults?
Experimentaldesignworkflow
Experimentaldesignworkflow
SeveraliteraFonstypicallyneededtorefine/understandtheresults!
ConsideraFonsforyourexperiment
• Treatments:whatareyoudoingtothecells?
ConsideraFonsforyourexperiment
• Controls:howwillyouknowifthetreatmenthasaneffect?
ConsideraFonsforyourexperiment
• Variables:whatareyoutesFng?
ConsideraFonsforyourexperiment
• Replicates:howwillyouknowtheresultsarereal?
OverviewofMod2
YourgoalistosynthesizetheinformaFon/dataintoasingle,coherentstory
geneexpression cellviabilitycancerbiology DNArepair
BackgroundInformaFon(publishedliterature)
ExperimentalData(yourresults!)
DetailsforResearcharFcle
• Submissiondue:Saturday,Apr20thby10pm
• Completedindividually• Onlyonedrag• Formaingguidelines:– PrepareusingWORD– Textandfiguresmaybeseparatedocuments
Howwillwecommunicateourscience?
• ResearcharFclestructureismoreformal– TitleandAbstract– IntroducFon– Methods– Results– Discussion
• Completesentencesandparagraphs• References
Howwillyoureportyourmethods?
Let’sfirstreviewthemethodslessonswelearnedfromourhomework…
1. Sub-secFons2. Levelofdetail3. Wordchoices
Methods:sub-secFons
• Usesub-secFonstogroupprocedures– IncludedescripFveFtles– Uselogical,ratherthanchronologicalorder
• Separatesub-secFonswithFtles– Brief,butspecific
• Includeanintroductorysentence– StatethepurposeorgoalofparFcularmethod/groupofprocedures
Methods:levelofdetail
Rememberthestepsyoudidn’tdo
• ForFmereasons,theteachingfacultycompletedsomeprocedures…– YoushouldsFllincludethese!– Detailsareprovidedonthewiki
DONOTWRITETHATTHESESTEPSWERECOMPLETEDBYTEACHINGFACULTY
Methods:wordchoices• thetubevs.thecelllysate– GivemoreinformaFve,specificinformaFon
• combinedormixedvs.digested– Bepreciseabouttheprocedureused
• cleanedvs.purifiedorisolated– UsethemorescienFficterminology
• inordertovs.to– Eliminateunnecessarywordiness
• avoidjargonanddefineallabbreviaFons
Considersentencestructure
“celllysatewaspreparedbyadding…andproteinswereseparatedusingSDS
polyacrylamidegelelectrophoresis(130Vfor45min)inTGSbuffer….”
1. PUTTHESUBJECTFIRST2. BESURETHESUBJECTANDTHEVERBMATCH
Whatareyourdata?
Howwillyouintroduceyourdata?
Howwillyoureportyourdata?
PROTIP:USESUB-SECTIONHEADERSTOSEPARATERESULTSTEXTACCORDINGTOFIGURES/DATA
AnexamplefromDietleinet.al.
RESULTSTEXT:
FIGURECAPTION:
Howwillyouinterpretyourdata?
ReporFngandinterpreFngyourdataRESULTS
1. Whatwastheoverallgoalofthesedata?– Stateconciselyasan
introductorysentence.2. Ifapplicable,whatwasthe
resultofyourcontrol?– Wasitexpected?
3. Whatwasyourresult?– Wasitexpected?
4. WhatdoesthismoFvateyoutodonext?– Specifically,what
experimentfollows?
DISCUSSION1. Whatevidencedoyou
havethatyourresultiscorrectorincorrect?– Howdoyourcontrols
supportyourdata?2. Insum,whatdoyourdata
suggestorindicate?– Doyourdatasupportyour
hypothesis?Why?3. WhatdoesthismoFvate
youtodonext?– Specifically,whatisthe
nextresearchquesFon?
Besuretoincorporatelecturematerial!
• Usetheanalysistoolsto‘do’somethinginteresFngwithyourdata– Howdothemethodscomparetooneanother?– Howdospecificgenes/funcFonschange?– Howdoyourresultsrelatetopublishedresearch?
• BecompleteinexplanaFonofdataanalysis– Whatdoesthemethodenableyoutest/conclude?Why?
Remembertotellastory!
• IntroducFonandDiscussionshouldmatch• ResultsshouldbeconnectedwithtransiFons• Discussionshould‘Fe’theresultstogetherintoacohesivetake-homemessage
• EffecFveredundancyservesapurpose– EachsecFoniscragedforaparFcularaudience