Northern blot: Diferenzas entre revisións

ProbesAs forsondas para northern blottingblot areestán composedcompostas ofde nucleicácidos acidsnucleicos withcunha asecuencia complementary[[complementariedade sequence(bioloxía tomolecular)|complementaria]] alla orunha partparte ofou thea RNAtodo ofo interestARN de interese, theypoden canser bede DNAADN, RNAARN, orou oligonucleotides[[oligonucleótido]]s withcun amínimo minimum ofde 25 complementary bases tocomplementarias thecoa targetsecuencia sequencediana.<ref name="Trayhurn1996" /> As sondas RNAde probesARN (riboprobesribosondas) thatque arese transcribedtranscriben in vitro arepoden ablesopartar touns withstandpasos morede rigorouslavado washingmáis stepsrigorosos, preventingo someque ofimpide theque backgroundapareza noisenos resultados parte do "ruído" de fondo.<ref name="Streit2009" /> Normalmente, Commonlypara cDNAfuncionar iscomo createdsonda withde labellednorthern primersblot forcréase the[[ADNc]] RNAcon sequence[[cebador]]es ofetiquetados interestpara toa actsecuencia asdo theARN probede in the northern blotinterese.<ref name=Liang1995>Liang, P. Pardee, A. B. (1995) Recent advances in differential display. Current Opinion Immunol. 7: 274–280.</ref> As Thesondas probesdeben mustetiquetarse becon labelled[[isótopo]]s either with radioactive isotopes[[redioactividade|radioactivos]] (³²P) orou withben con [[chemiluminescencequimioluminescencia]], inna cal os whichencimas [[alkalinefosfatase phosphatasealcalina]] orou [[horseradish peroxidase do ravo picante]] break(''Armoracia downrusticana'') chemiluminescentdegradan substratesos producingsubstratos aquimioluminescentes detectableproducindo emissionunha ofemisión lightde luz detectable.<ref name=Engler-Blum1993>{{cite journal | last1 = Engler-Blum | first1 = G. | last2 = Meier | first2 = M. | last3 = Frank | first3 = J. | last4 = Muller | first4 = G. A. | year = 1993 | title = Reduction of Background Problems in Nonradioactive Northern and Southern Blot Analysis Enables Higher Sensitivity Than 32P-Based Hybridizations | url = | journal = Anal. Biochem | volume = 210 | issue = 2| pages = 235–244 | doi = 10.1006/abio.1993.1189 | pmid = 7685563 }}</ref> A Theetiquetaxe chemiluminescentquimiolunimescente labellingpode canfacerse occurde in twodous waysxeitos: eitherpode theunirse probeo isencima attachedá to the enzymesonda, orou thepode probeetiquetarse isa labelledsonda withcun a ligand[[ligando]] (e.g.por exemplo, [[biotinbiotina]]), forpara whicho thecal antibodyse une ao encima un [[anticorpo]] (e.g.por exemplo, [[avidinavidina]] orou [[streptavidinestreptavidina]]) is attached to the enzyme.<ref name="Streit2009" /> Cunha película de [[raios X-ray]] filmpode candetectarse detecttanto bothos thesinais radioactiveradioactivos andcoma chemiluminescentos signalsquimioluminescentes, ande manymoitos researchersinvestigadores preferprefiren theos chemiluminescentsinais signalsquimioluminescentes becauseporque theyson aremáis fasterrápidos, moremáis sensitivesensibles, ande reducereducen theos healthperigos hazardspara thata gosaúde alongaociados withcoas radioactivemarcaxes labelsradioactivas.<ref name="Engler-Blum1993" /> Unha Themesma samemembrana membranepode cansondarse beata probedcinco upveces tosen fiveque timesse withoutproduza aunha significantperda loss ofsignificativa thede targetARN RNAdiana.<ref name="Yang1993" />