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