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Inkokeli

I-RNA ende engeyiyo ikhowudi, i-lncRNA yi-RNA engabhaliyo enobude obungaphezulu kwama-nucleotides angama-200, ngokubanzi phakathi kwe-200-100000 nt.I-lncRNA ilawula ukubonakaliswa kofuzo kumanqanaba e-epigenetic, ukukhutshelwa kunye nasemva kokubhala, kwaye ithatha inxaxheba ekuthuliseni i-chromosome ye-X, ukuprintwa kwe-genome kunye nokuguqulwa kwe-chromatin, ukusebenza kwe-transcription, ukuphazamiseka kokubhala, uthutho lwenyukliya, ukulawulwa komjikelo weseli, umgaqo wokwahlulahlula iiseli, kunye neziphumo zembuyekezo yedosi ezininzi kunye neziphumo ezinxulumene nembuyekezo yedosi. uthintelo lwezifo zabantu, kwaye ngoku yenye yeendawo ezishushu kwinkalo ye-biomedicine.

01  Iintlobo kunye neempawu ze-LncRNA

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I-lncRNA yohlulwe kwiindidi ezininzi ngokwemithombo eyahlukeneyo yokwakheka kwayo, njengoko kubonisiwe kulo mfanekiso ungentla.Inezi mpawu zilandelayo:

1. I-lncRNAs zidla ngokuba nde, zinentetho eguquguqukayo kunye neendlela ezahlukeneyo zokuhlanganisa ngexesha lokwahlula.
2. Xa kuthelekiswa nohlobo lwekhowudi, i-lncRNA idla ngokuba nenqanaba elisezantsi lokubonisa
3. Uninzi lwe-lncRNAs luneenkcukacha ezicacileyo zexesha kunye nesithuba kwinkqubo yokwahlulwa kwezicubu kunye nophuhliso.
4. Kukho ukubonakaliswa kweempawu kwiithumba kunye nezinye izifo
5. Iindawo ezisezantsi ze-lncRNA zihlukeneyo
6. Ulandelelwano luphantsi kulondolozo, kodwa umsebenzi unomlinganiselo othile wolondolozo, njl.

02 Imibuzo Ebuzwa Rhoqo malunga ne-LncRNA Reverse Transcription

Ngenxa yomxholo ophantsi we-lncRNA kwiiseli, ubude obude kunye nesakhiwo somgangatho ophezulu, i-RNA enjalo ihlala inezakhiwo eziyinkimbinkimbi ezifana ne-stem loops okanye i-hairpins, kwaye i-RT-qPCR ixhomekeke kwiingxaki zokungaphumeleli ukuguqulelwa kwe-reverse okanye ukusebenza okuphantsi.

03 Isisombululo se-LncRNA sokuguqulela umva

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Kumgaqo osembindini, inkqubo yeentsholongwane ze-RNA zisebenzisa eyazo i-reverse transcriptase ukwenza i-DNA isebenzisa i-RNA njengethempleyithi ibizwa ngokuba yi-reverse transcription.Inkqubo yokusebenzisa i-RNA virus reverse transcriptase ukudibanisa itemplate ye-cDNA kwi-vitro ibizwa ngokuba yi-reverse transcription.Izinto eziphambili ezibandakanyiweyo kwi-reverse transcription system: i-template ye-RNA, i-reverse transcriptase, i-primers kunye namanye amacandelo.

Itemplate ye-1.RNA

Impembelelo yethemplate ye-RNA kwi-reverse transcription ibonakala kwisakhiwo sayo, ubunyulu kunye nokunyaniseka.Okukhona kuphezulu ukusulungeka kunye nokuthembeka kwe-RNA ekhutshiweyo, kokukhona kuphezulu ukusebenza kakuhle kokukhutshelwa umva kunye nokuchaneka ngakumbi iziphumo zobungakanani obulandelayo.I-RNA ehlanjululwe zizinto ezisetyenziswa ngokuqhelekileyo ze-RNA zokutsalwa zihlala ziqulethe iintsalela zotywala kunye neetyuwa ze-guanidine, ezinempembelelo enamandla yokuthintela kwi-reverse transcriptase.

2.Reverse transcriptase

I-Reverse transcriptase inefuthe eliphambili kuyo yonke inkqubo yokukhuphela umva.Ubushushu obufanelekileyo be-reverse transcriptase esetyenziswa kwiilabhoratri ezininzi ngokuqhelekileyo ngama-42°C.Enyanisweni, ukuvulwa kwesakhiwo se-RNA esiphezulu kufuna ukushisa okuphezulu, kwaye iimfuno ze-reverse transcriptase ziphezulu.

3.Isiqalo

Iiprayimari zokukhuphela umva ziquka iiprimers zegene-specific, ii-random primers kunye ne-Oligo dT primers.Uninzi lwe-lncRNA alunayo imisila ye-polyA, kwaye kubalulekile ukufanisa ngokufanelekileyo i-random primers kunye ne-Oligo dT.

4.Amanye amacandelo

Kwimbono yokuba i-RNA ilula kakhulu ukuthotywa, ukuqaliswa kwamacandelo kwinkqubo yokukhuphela umva kunokuncitshiswa, okunokuthi kuthintele ngokufanelekileyo ukungena kwe-RNase kunye nokuqinisekisa inkqubela phambili egudileyo yokukhutshelwa umva.

Foregeneifumana kwithemplethi ye-RNA, ukubuyisela umva ukhutshelwe kwi-qPCR

Khupha ngokugqibeleleyo ukufunyaniswa kwakho kwe-lncRNA

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Izilwanyana zizonke RNA IuxoloIkhithi (enekholamu yokususa i-gDNA)

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ISeli iyonke iRNA IuxoloIkhithi (enekholamu yokususa i-gDNA)

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I-Lnc-RT Hero TM I(Nge-gDNase)(I-Super Premix ye-cDNA ye-first-strand synthesis esuka kwi-lncRNA)

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Ixesha lokwenyani PCR EasyTM-SYBR Green I

Aizinto eziluncedo ofUtsalo lwe-RNA:

Ikiti isebenzisa iteknoloji ye-RNA yesizukulwana sesithathu, akukho mfuneko yokusebenzisa i-DNase.

Imizuzu eyi-11 ukusuka kwi-96, 24, 12, kunye ne-6-well plate cultured cells, i-high-effective extraction ye-gDNA-free, high-purity, high-quality total cell RNA.

Izinto eziluncedo zokukhutshelwa umva kwe-lncRNA:

Inkqubo yokubhala umva eyenzelwe i-LncRNA ukususa ngokukhawuleza ungcoliseko lwe-genomic DNA.

Inkqubo yokukhutshelwa umva inozinzo oluphezulu lwe-thermal kwaye isenomsebenzi oncomekayo wokukhuphela umva kwi-50°C.

Izifanekiso ezinomxholo ophezulu we-GC kunye nesakhiwo sesibini esiyinkimbinkimbi sinokutshintshwa ngokusebenza okuphezulu.

Iingeneloof qI-PCR:

Isiqalo esishushu i-Foregene Taq Polymerase inomgangatho ophezulu wokukhulisa umvandedwa, uvakalelo oluphezulu lokukhulisa, kunye nokucaciswa okuphezulu kokukhulisa

I-PCR yokwenene yoMxube olula yenza ukuba i-SYBR Green ndibe novakalelo oluphezulu lokubona, kwaye ubunzulu bayo be-fluorescence buphindwe ka-3-5 kwiimveliso ezifanayo, ezinokuhlangabezana neemfuno zeentlobo ezahlukeneyo zovavanyo lobungakanani be-fluorescence.


Ixesha lokuposa: Jul-23-2021