Ƙayyadaddun ganowa
A mafi yawan lokuta, manufar ƙira na farko shine don haɓaka ƙayyadaddun PCR.Ana ƙayyade wannan ta ƙarin ko žasa tasirin da ake iya faɗi na yawancin masu canji.Ɗaya mai mahimmanci mai mahimmanci shine jerin a ƙarshen 3' na farko.
Mahimmanci, ƙididdigar PCR da aka tsara don ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai sun fi dacewa don kula da babban inganci akan kewayon ƙarfi mai fa'ida, saboda ƙididdigar ba ta samar da takamaiman samfuran haɓakawa ba, don haka yin gasa tare da reagents na PCR ko hana babban haɓakawa.
Tabbas, a wasu lokuta, ƙayyadaddun ba shine mafi mahimmanci ba, misali, lokacin da makasudin shine ƙididdige alaƙa da alaƙa amma ana buƙatar ƙwayoyin cuta daban-daban, ƙira na musamman, haɓakawa da matakan tabbatarwa.
Tsarin narkewa shine daidaitaccen hanya don tantance ƙayyadaddun abubuwan amplicons, aƙalla dangane da ko za a haɓaka manufa ɗaya.Duk da haka, dole ne a jaddada cewa magudanar narkewa na iya zama ɓatarwa saboda, alal misali, za a iya shafa su ta hanyar haɗakar da abubuwan da suka dace da ƙananan ƙananan ƙananan ƙira.
P5 |Tsarin narkewa yana nuna sauye-sauyen Tm da aka samu daga ganowa biyu na adadi daban-daban na DNA guda biyu.
A. A mafi girma taro (ad)), babu bayyanannen dimer na farko bayan an kammala ma'aunin qPCR.Yayin da tarin samfuri ya ragu zuwa kwafi 50 (e), samfurin da ba takamaiman ba ya fara bayyana kuma ya zama samfuri ɗaya kaɗai a mafi ƙanƙanci (f).
B. Gwajin ya rubuta Tms iri ɗaya a duk maƙasudin maƙasudi, kuma babu bayyananniyar dimer na farko ko da a mafi ƙanƙanta taro (kofi 5).Lokacin amfani da waɗannan hanyoyin ganowa guda biyu, ba a sami samfuran ƙarawa a cikin NTCs ba.
P5 yana nuna maɓallan narkar da da aka samu tare da samfurori wanda samfurin ya kasance a wurare daban-daban.P 5a yana nuna cewa a mafi ƙasƙanci mafi ƙasƙanci guda biyu, Tms na samfuran haɓakawa na musamman waɗanda aka samar sun kasance ƙasa da na takamaiman amplicons.
Babu shakka, wannan hanyar ganowa ba za a iya dogaro da ita don gano maƙasudin da ke wanzuwa a cikin ƙananan ƙira ba.
Abin sha'awa, NTCs, watau, samfuran da ba su da DNA kwata-kwata, ba su yi rikodin (marasa takamaiman) samfuran haɓakawa ba, yana nuna cewa asalin halittar DNA na iya shiga cikin haɓakawa/polymerization marasa takamaiman.
Wasu lokuta ba za a iya gyara irin waɗannan na'urori na baya da haɓakawa ba, amma sau da yawa yana yiwuwa a tsara hanyar ganowa wanda ba shi da ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun samfurin da kuma NTC (P 5b).
Anan, har ma da yin rikodin haɓakar ƙaddamar da niyya tare da Cq na 35 zai haifar da ƙayyadaddun yanayin narkar da.Hakazalika, NTCs ba su nuna alamun haɓakawa na musamman ba.Wani lokaci, halayen ganowa na iya dogara ga mahaifiyar giya, kuma kawai ƙarawa mara iyaka kawai ana iya ganowa a cikin wasu abubuwan da aka haɗa, waɗanda ƙila suna da alaƙa da ma'aunin Mg2+ daban-daban.
Karfin ganowa
Haɓaka Ta mataki ne mai fa'ida a cikin ingantaccen tabbaci da ingantaccen tsarin gano qPCR.Yana ba da nuni kai tsaye na ƙarfin firikwensin da aka saita ta hanyar nuna zafin jiki (ko kewayon zafin jiki) wanda ke samar da mafi ƙarancin Cq ba tare da haɓaka NTC ba.
Bambanci sau biyu zuwa hudu a hankali na iya zama ba mahimmanci ga mutanen da ke da babban maganganun mRNA ba, amma don gwaje-gwajen bincike, yana iya nufin bambanci tsakanin sakamako mai kyau da na ƙarya.
Kaddarorin Ta na qPCR na farko na iya bambanta sosai.Wasu gwaje-gwajen ba su da ƙarfi sosai, kuma idan ba a yi su a ƙarƙashin mafi kyawun darajar Ta na firam ɗin ba, za su rushe da sauri.
Wannan yana da mahimmanci saboda irin wannan ganowa sau da yawa yana da matsala a cikin ainihin duniya, kuma tsarkin samfurin, ƙaddamar da DNA, ko kasancewar wasu DNA bazai zama mafi kyau ba.
Bugu da kari, lambar kwafin manufa na iya bambanta a cikin kewayo mai fadi, kuma reagents, kayan aikin filastik, ko kayan aiki na iya bambanta da waɗanda aka yi amfani da su yayin kafa gwajin.
P6|Ma'aunin zafin jiki yana nuna bambancin ƙarfin gano PCR.
A. Yi amfani da Bioline's Sensifast SYBR mastermix (lambar kundin BIO-98050) don yin PCR akan cDNA da aka shirya daga kwakwalwar ɗan adam RNA.
B. Yi amfani da kayan aikin Bio-Rad's CFX qPCR don yin rikodin taswirar haɓakawa da narkarwar apalene (NM_033207, F: GCCATGGAGGAAAGTGACAGACC, R: CTCATGTGTGGTGATCTCCTAGG).
C. Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙaƙwalwa na ACSBG1 (NM_015162.4, F: CTACCTTCCGGCACCACTGG, R: GTCCACGTGATATTGCTCTGACTCAG).
D. Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwaƙwalwa na GFAP (NM_002055.5, F: TGGAGAGGAAGATTGAGTCGCTGG, R: CGAACCTCCTCCTCGTGATCTTC).
E. Cqs da aka rubuta a yanayin zafi daban-daban, suna nuna bambanci a cikin Cq da aka yi rikodin ƙarƙashin madaidaicin zafin jiki na 7C.
P 6 yana nuna sakamako na yau da kullun na gwajin da ba a so, inda aka yi qPCR ta amfani da gradient Tas tsakanin 59C da 67C (P 6a), ta yin amfani da na'urori masu mahimmanci don takamaiman ƙwayoyin mutum guda uku.
Ana iya gani daga jadawali na ƙarawa cewa Opalin primers sun yi nisa da manufa domin mafi kyawun kewayon Ta suna da kunkuntar (Hoto na 6b), wato Cqs sun tarwatse sosai, wanda ya haifar da Cqs sosai idan aka kwatanta da mafi kyawun Cqs Low.
Wannan hanyar ganowa ba ta da kwanciyar hankali kuma tana iya haifar da haɓakawa mafi inganci.Saboda haka, ya kamata a sake fasalin wannan nau'i-nau'i biyu.Bugu da ƙari, ƙididdiga na narkewa (inset) ya nuna cewa ƙayyadaddun wannan hanyar ganowa na iya zama matsala, saboda yanayin narkewa na kowane Ta ya bambanta.
Hanyar gano ACSBG1 da aka nuna a cikin P 6c ta fi ƙarfi fiye da hanyar gano Opalin a sama, amma har yanzu yana da nisa daga manufa, kuma yana yiwuwa a iya inganta shi.
Duk da haka, muna jaddada cewa babu wata hanyar da ta dace tsakanin ƙarfi da ƙayyadaddun ƙayyadaddun bayanai, saboda yanayin da aka samar ta wannan hanyar ganowa yana nuna ƙimar kololuwa ɗaya a cikin duk Tas (saka).
A gefe guda kuma, gwajin ƙarfin ƙarfi ya fi haƙuri, yana samar da irin wannan Cqs a cikin kewayon Tas, kamar yadda a cikin gwajin GFAP da aka nuna a P 6d.
Bambanci a cikin Cqs da aka samu a cikin kewayon ma'aunin ma'aunin Celsius 8 bai wuce 1 ba, kuma madaidaicin narkar da (saka) yana tabbatar da halayen ganowa a cikin wannan kewayon zafin jiki.Yana da kyau a lura cewa Tas da aka ƙididdigewa da ainihin kewayon Ta na iya bambanta sosai.
Akwai jagorori da yawa da aka tsara don taimakawa masu bincike suna tsara ingantattun kamfaki, yawancinsu suna bisa doguwar ƙa'idodi da yawa da aka biya su na Firayim.Yawancin lokaci ana ba da shawarar haɗa G ko C a ƙarshen 3' da sansanonin G ko C guda biyu (ƙuƙuman GC), amma bai wuce biyu na tushe 5 na ƙarshe ba.
A aikace, waɗannan dokoki na iya jagorantar masu bincike, amma ba lallai ba ne su kasance daidai a kowane yanayi.
P7 |Ƙarshen 3' na farko yana da ɗan tasiri akan ƙayyadaddun ƙayyadaddun aiki ko inganci.
A. Matsayin masu farawa don HIF-1a (NM_181054.2) na ɗan adam.
B. Yi amfani da Agilent Brilliant III SYBR Green uwar barasa (Cat. No. 600882) don haɓaka abubuwan gwaji shida.
C. Girman jadawali da lanƙwan narkewa da aka rubuta ta kayan aikin qPCR na Bio-Rad na CFX da 3′end primers.Ana nuna NTC da ja.
D. Cqs rikodin kowane abu na gwaji
Misali, sakamakon a cikin P 7 ya saba wa ka'idar 3'end.Duk ƙirar ƙira tana samar da sakamako iri ɗaya, tare da haɗaɗɗun firamare guda biyu kawai waɗanda ke haifar da haɓakawa marasa takamaiman a cikin NTC.
Koyaya, ba za mu iya tallafawa tasirin shirin GC ba, saboda a cikin wannan yanayin, yin amfani da A ko T azaman matsakaicin tushe 30 baya rage takamaiman.
Gwajin C, inda F na farko ya ƙare a GGCC, ya yi rikodin Cqs a cikin NTCs, yana nuna cewa mutum zai so ya guje wa waɗannan jeri a ƙarshen 30.Muna jaddada cewa hanya ɗaya tilo don tantance mafi kyawun jerin 3′ƙar ƙarshen na farkon biyu shine a kimanta wasu ƴan takara da gwaji.
Ingantaccen haɓakawa
Mahimmanci, kodayake ganowar PCR ba takamaiman ba ba zai taɓa zama takamaiman ba, ana iya daidaita haɓakar haɓakawa da haɓaka ta hanyoyi daban-daban ta hanyar canza enzyme, giya mai uwa, ƙari, da yanayin hawan keke.
Don kimanta ingancin gano PCR, yana da kyau a yi amfani da serial dilution na 10 ko 5 sau da manufa nucleic acid, wato, "daidaitaccen hanyar lankwasa".
Idan ana amfani da amplicons na PCR ko maƙasudin DNA na roba don samar da daidaitaccen lanƙwasa, ya kamata a gauraya serial dilutions na waɗannan maƙasudi tare da adadin DNA na baya (kamar DNA na genomic).
P8 |Dilution curve don kimanta ingancin PCR.
A. Yi amfani da firamare don HIF-1: F: AAGAACTTTTAGGCCGCTCA da R: TGTCCTGTGGTGACTTGTCC da Agilent's Brilliant III SYBR Green mastermix (lambar kasida 600882) don PCR da yanayin yanayin narkewa.
B. 100 ng RNA an juyar da shi, an diluted sau 2, kuma an diluted samfuran cDNA a jere sau 5 zuwa 1 ng DNA na ɗan adam.Ana nuna yanayin narkewa a cikin sa.
C. Halin RT, dilution, da dilution serial an maimaita su don samfurin cDNA na biyu, kuma sakamakon ya kasance iri ɗaya.
P 8 yana nuna ma'auni guda biyu, ta yin amfani da hanyar ganowa iri ɗaya akan samfuran cDNA guda biyu daban-daban, sakamakon shine daidaitattun daidaitattun, kusan 100%, kuma darajar R2 kuma tana kama da haka, wato, matakin dacewa tsakanin bayanan gwaji da layin regression ko bayanan Degree na layi.
Matsakaicin ma'auni guda biyu suna kama da juna, amma ba daidai suke ba.Idan manufar ita ce ƙididdige maƙasudin daidai, dole ne a lura cewa ba za a yarda da samar da lissafin kwafi ba tare da bayyana rashin tabbas ba.
P9 |Rashin tabbas na auna da ke da alaƙa da ƙididdigewa ta amfani da daidaitaccen lanƙwasa.
A. Yi amfani da firamare don GAPDH (NM_002046) don aiwatar da PCR da yanayin lanƙwasa.F: ACAGTTGCCATGTAGACC da R: TAACTGGTTGAGCACAGG da Bioline's Sensifast SYBR mastermix (lambar kundin BIO-98050).
B. Taswirar haɓakawa, lanƙwan narkewa da daidaitaccen lanƙwasa da aka rubuta tare da kayan aikin qPCR na Bio-Rad na CFX.
C. Madaidaicin jadawali mai lankwasa da 95% tazarar amincewa (CI).
D. Lambar kwafin da tazarar amincewa 95% na ƙimar Cq guda uku waɗanda aka samo daga madaidaicin dilution.
P 9 yana nuna cewa don ingantacciyar gwaji, bambance-bambancen da ke tattare da ma'auni guda ɗaya shine kusan sau 2 (95% tazarar amincewa, ƙarami zuwa matsakaicin), wanda zai iya zama mafi ƙarancin sauye-sauyen da za'a iya sa ran.
Samfura mai alaƙa:
Lokacin aikawa: Satumba-30-2021
