Page 14 - Demo

33Molecularparametersassays33aRNAExtractionTheAmbion®RiboPureTMkitwasusedtoisolateribonucleicacid(RNA)fromlivertissuesForthispurpose100mgoflivertissuewereobtainedandwerelaterhomogenisedinthepresenceof TRIReagent® with the TissueLyser® TRIReagent®containsbothphenolandguanidinethiocyanatewhichlysethecellsandinactivatenucleasesinorder topreventnucleicaciddegradationThen thismixture wascentrifuged to separateall theinsolublecomponentsThesupernatantwascollectedandbromochloropropane(BCP)wasaddedBCPproducestheformationoftwophasesaqueousphaseandorganicphaseRNAwillstayintheaqueousphasewhiledesoxyribonucleicacid(DNA)andproteinsremainintheinterphaseortheorganicphaseTheaqueousphasewassubsequentlyintroducedinaseriesofcolumns topurifyandextract theRNA which waslaterquantifiedin theNanodrop® Thenanagarosegelelectrophoresis wasperformed to verify theintegrityofRNA(Figure5)ToensurethetotaleliminationofDNAfromthesamplethe Ambion® TURBODNA-freeTMkitwasemployed Thiskitcontainsdeoxyribonucleases(DNases)thatdegradetheDNApresentThenasa way toconfirm thatanycontaminationofDNAhadbeenremovedapolymerasechainreaction(PCR)usingRPS29(ribosomalproteinS29)primersandthesubsequentagarosegelelectrophoresiswereperformedTheoutcomewasasexpectedonlythepositivecontrolshowedanitidresultandnobandsappearedin thesamplescontainingRNA treated withDNaseconfirming theabsenceofDNAcontaminant33bReverseTranscription-PCR(RT-PCR)AfterthepurificationstepwithDNasethesampleonlycontainedRNAInorder toquantify theamountofmessengerRNA(mRNA)inthesamplesthroughrealtimePCRitispreviouslyrequired toconvert themRNAintocomplementaryDNA(cDNA) Withthisaimareverse transcriptasepolymerasechainreaction(RT-PCR)wascarriedoutusingtheSuperScriptTMIIReverseTranscriptase32bPlasmatriglyceridesdeterminationIt wasperformed withanenzymaticcolorimetricend-pointassay,usingaSpinreact(Spain)kit32cHDLcholesteroldeterminationTheobjectivewasthedeterminationofHigh-densityLipoprotein(HDL)cholesterollevelsFor thispurposea two-stepprocesswascarriedout32caPrecipitationofnon-HDLcholesterollipoproteinsPhosphotungsticacidincombinationwithmagnesiumchlorideisable toprecipitateapoBcontaininglipoproteins(chylomicronsVLDLandLow-densityLipoproteins(LDL))ThekitusedwasfromSpinreactSpainThevolumesemployedwere100μLofsampleand10μLHDLc-P(theprecipitatingreagent)Aftera10-minuteincubationatroomtemperaturethesampleswerecentrifugedat12000rpmfor30minutesatroomtemperatureThesupernatantwasused forsubsequentHDL-cholesterollevelsdetermination32cbHDLcholesteroldeterminationThedeterminationofHDLcholesterollevelswasperformedwithanenzymaticcolorimetricend-pointassay,usingaSpinreact(Spain)kitItisbasedon thedetectionat 505nmofacolouredcompound(quinoneimine) thatisgenerated fromcholesterolaftersequentialenzymaticreactions Thecalculationofnon-HDLcholesterolwasobtainedfromthedifferenceoftotalcholesterolconcentrationminusHDLcholesterolconcentration32dCoronaryRiskIndex(CRI)TheCRIiscalculatedfromtotalcholesterolandHDL-cholesteroldataTheformulais𝑇𝑜𝑡𝑎𝑙𝑐ℎ𝑜𝑙𝑒𝑠𝑡𝑒𝑟𝑜𝑙𝐶𝑅𝐼=𝐻𝐷𝐿𝑐ℎ𝑜𝑙𝑒𝑠𝑡𝑒𝑟𝑜𝑙32e AtherogenicIndexofPlasma(AIP)The AIPiscalculated from triglycerideandHDL-cholesteroldataTheformulais𝑇𝑟𝑖𝑔𝑙𝑦𝑐𝑒𝑟𝑖𝑑𝑒𝑠API=log𝐻𝐷𝐿𝑐ℎ𝑜𝑙𝑒𝑠𝑡𝑒𝑟𝑜𝑙262ANALESRANFwwwanalesranfcomLaingestamaternade fructosamodulaelmetabolismodelcolesterolenrespuestaaunadietaoccidentalenladescendenciaPalomaLópezLaizElenaFausteetalAnRealAcadFarmVol88Nº3(2022)·pp257-275()Figure 5 AgarosegelelectrophoresisofRNAextraction The twomore visiblebandscorrespondtothe28Sand18SribosomalRNA