InterpretingNanodrop(Spectrophotometric)Results

FoundationofSpectrophotometry:TheBeerLambertLawwhatdoesitmean?
A = cl
WhereA=absorbance,=extinctioncoefficient,c=concentrationandl=pathlength.
TheBeerLambertlawdrawsadirectcorrelationbetweenabsorbanceandconcentration.
Whilenucleicacidsabsorbatmanywavelengths,theyhaveapeakabsorbanceofUVlightat
260nm.Thus,theamountoflightabsorbedinthisregioncanbeusedtodeterminethe
concentrationofRNAorDNAinsolutionbyapplyingtheBeerLambertlaw.However,the
BeerLambertequationisonlylinearforabsorbancesbetween0.1and1.0.Thistranslatesto
concentrationsbetween10.0ng/uLand3700ng/uLwhenusingtheNanodropND1000.
Samplesoutsideofthisrangeshouldbedrieddownordilutedtoproducemoreaccurate
spectrophotometryresults.
Absorbanceat260nm
NucleicacidsabsorbUVlightat260nmduetothearomaticbasemoietieswithintheir
structure.Purines(thymine,cytosineanduracil)andpyrimidines(adenineandguanine)
bothhavepeakabsorbancesat260nm,thusmakingitthestandardforquantitating
nucleicacidsamples.
Absorbanceat280nm
The280nmabsorbanceismeasuredbecausethisistypicallywhereproteinsandphenolic
compoundshaveastrongabsorbance.Aromaticaminoacidsidechains(tryptophan,
phenylalanine,tyrosineandhistidine)withinproteinsareresponsibleforthisabsorbance.
Similarly,thearomaticityofphenolgroupsoforganiccompoundsabsorbsstronglynear
280nm.
Absorbanceat230nm
Many organic compounds have strong absorbances at around 225 nm. In addition to
phenol,TRIzol,andchaotropicsalts,thepeptidebondsinproteinsabsorblightbetween
200and230nm.
A260/280ratio
TheA260/280ratioisgenerallyusedtodetermineproteincontaminationofanucleicacid
sample.ThearomaticproteinshaveastrongUVabsorbanceat280nm.ForpureRNAand
DNA,A260/280ratiosshouldbesomewherearound2.1and1.8,respectively.Alowerratio
indicatesthesampleisproteincontaminated.Thepresenceofproteincontaminationmay
haveaneffectondownstreamapplicationsthatusethenucleicacidsamples.

A260/230ratio

TheA260/230ratioindicatesthepresenceoforganiccontaminants,suchas(butnotlimited
to):phenol,TRIzol,chaotropicsaltsandotheraromaticcompounds.Sampleswith260/230
ratiosbelow1.8areconsideredtohaveasignificantamountofthesecontaminantsthatwill
interferewithdownstreamapplications.Thisisespeciallytrueforreversetranscription.Ina
puresample,theA260/230shouldbecloseto2.0

RealWorldExamples:

TheabsorbancespectrumoftheRNAsamplebelowindicatesahighpuritywithclosetoideal
A260/280andA260/230ratios.Alsonotethattheconcentrationiswithinthereliablerangeof
theNanodropND1000.

Interpretation:thissampleissuitablefordownstreamapplications.
TheRNAsamplebelowhasagoodA260/280ratio,indicatingnopresenceofprotein
contaminants,however,theA260/230ratioof1.29issignificantlylowerthan2indicating
somesortoforganiccontaminantspresentinthesample.Notehowthereisahighshoulder
at220nm(comparetothepuresampleabove)andtheresabulgeonthepeakshoulderat
270nm,alsoindicativeofcontamination.Typically,thecontaminationiswithreagentsused
intheisolationprodecure:TRIzol,phenol,orchaotropicsalt(guanidiniumisothiocyanate,aka
GITC).

Absorbanceat220and270by
contamination

Interpretation:thissampleshouldberepurifiedonacolumn(orsomeothermethod).

The RNA sample below has low A260/280 and A260/230 ratios. Additionally, themaximum
peakisat270nm.Thiscurveistypicalofahighconcentrationofphenol(orGITC)inthesample
asaresultofTRIzolRNAisolationprotocolswithpoorremovalofphenolduringtheorganic
phaseseparations.Noteagain,thehighshoulderofthecurveat220nmandthattheactual
nucleicacidpeakat260nmappearsasasmallshoulderinthemuchlargerphenolpeakat
270nm.

ThisistheRNAabsorbance,
dwarfed by the contamination.

Intepretation:duetothelowpurity,thissampleshouldbereisolatedonacolumn(ieQiagen
RNeasykit).NotethattheconcentrationofRNAreturnedbythespectrophotometerisnotat
allreliableduetothecontamination.

BelowisanRNAsamplewithaverylowconcentration.Becauseofthelowconcentration,itis
difficulttoassessthepurityofthesamplebyanalyzingtheA260/280andA260/230ratios.This
samplemayactuallybegood,butitcannotbeassessedbytheNanoDropbecauseitisoutside
thelowestconcentrationtheNanoDropisdesignedtomeasure.Amoresensitivemethodsuch
asanAgilentBiolanalyzerorQubitanalysisisrecommended.

Interpretation:UseamoresensitivemethodtomeasuretheRNAconcentration

Finally,keepinmindthatnucleicacidsabsorbat260.Justbecauseyouhaveabsorbanceat260
nmdoesnotmeanthatyourRNAisintact,itcouldbecompletelydegraded,oritcouldactually
beDNA(alwaysdotheoptionaloncolumnDNAsetreatment).