Ketapele

RNA e telele e sa khouteng, lncRNA ke RNA e sa khoute e nang le bolelele bo fetang li-nucleotide tse 200, hangata lipakeng tsa 200-100000 nt.lncRNA e laola liphatsa tsa lefutso maemong a epigenetic, transcript le post-transcriptal, hape e nka karolo ho khutsiseng chromosome ea X, khatiso ea genome le phetoho ea chromatin, ts'ebetso ea transcript, tšitiso ea transcript, lipalangoang tsa nyutlelie, taolo ea potoloho ea lisele, taolo ea karohano ea lisele, le lits'ebetso tse ling tsa puseletso tse amanang le lethal dose. thibelo ea mafu a batho, 'me hajoale ke e' ngoe ea libaka tse chesang haholo lefapheng la biomedicine.

01  Mefuta le litšobotsi tsa LncRNA

lncRNA e arotsoe ka mefuta e mengata ho ea ka mehloli e fapaneng ea sebopeho sa eona, joalokaha ho bontšitsoe setšoantšong se ka holimo.E na le litšobotsi tse latelang:

1. Li-lncRNA hangata li telele, li na le polelo e matla le mekhoa e fapaneng ea ho kopanya nakong ea phapang.
2. Ha ho bapisoa le liphatsa tsa lefutso tsa khouto, lncRNA hangata e na le boemo bo tlase ba polelo
3. Li-lncRNA tse ngata li na le polelo e hlakileng ea nakoana le ea sebaka molemong oa ho arola le nts'etsopele ea lisele.
4. Ho na le lipolelo tse khethollang lihlahala le mafu a mang
5. Libaka tse nyenyane tsa lncRNA li fapane
6. Tatelano e tlase ka paballo, empa mosebetsi o na le tekanyo e itseng ea paballo, joalo-joalo.

02 Lipotso tse botsoang khafetsa ka LncRNA Reverse Transcription

Ka lebaka la litaba tse tlase tsa lncRNA liseleng, bolelele bo bolelele le sebopeho sa boemo bo holimo, RNA e joalo hangata e na le meaho e rarahaneng joalo ka li-stem loops kapa li-hairpins, 'me RT-qPCR e na le mathata a ho ngola reverse kapa ho se sebetse hantle.

03 LncRNA reverse transcript solution

Molaong o bohareng, ts'ebetso ea livaerase tsa RNA tse sebelisang reverse transcriptase ea tsona ho kopanya DNA ka RNA joalo ka template e bitsoa reverse transcript.Mokhoa oa ho sebelisa RNA virus reverse transcriptase ho kopanya thempleite ea cDNA in vitro e bitsoa reverse transcription.Lintlha tse ka sehloohong tse kenyellelitsoeng tsamaisong ea reverse transcript: template ea RNA, reverse transcriptase, primers le likarolo tse ling.

1.RNA setšoantšo

Tšusumetso ea template ea RNA ho reverse transcript e bonahala ka sebopeho sa eona, bohloeki le botšepehi.Ha ho hloeka le ho ts'epahala ha RNA e ntšitsoeng holimo, ho etsa hore ho be le bokhoni bo phahameng ba ho ngoloa ka morao 'me liphetho tse latelang tsa palo li nepahetse.RNA e hloekisitsoeng ka li-reagents tse sebelisoang ka tloaelo tsa RNA hangata e na le mesaletsa ea joala le letsoai la guanidine, tse nang le phello e matla ea ho thibela ho fetisisa reverse transcriptase.

2. Reverse transcriptase

Reverse transcriptase e na le tshusumetso e kgolo tsamaisong yohle ya dingolwa tsa reverse.Thempereichara e loketseng bakeng sa reverse transcriptase e sebelisoang lilaboratoring tse ngata hangata ke 42°C.Ha e le hantle, ho buloa ha sebopeho sa boemo bo phahameng ba RNA ho hloka mocheso o phahameng, 'me litlhoko tsa reverse transcriptase li phahame.

3.Se qalang

Li-primers tsa reverse transcript li kenyelletsa li-primers tse ikhethileng tsa lefutso, li-primers le li-primer tsa Oligo dT.Li-lncRNA tse ngata ha li na mehatla ea polyA, 'me ho bohlokoa ho tsamaisana hantle le li-primers le Oligo dT.

4.Likarolo tse ling

Ka lebaka la 'nete ea hore RNA e bonolo haholo ho e theola, ho hlahisoa ha likarolo tsa mokhoa oa ho fetolela ka morao ho ka fokotsoa, ​​​​e leng se ka thibelang ho kena ha RNase ka katleho le ho netefatsa tsoelo-pele e ntle ea ho fetolela ka morao.

Foregenee fumana ho tsoa ho template ea RNA, e khutlisetsa morao ho qPCR

Phehella lncRNA ea hau ka botlalo

Kakaretso ea Liphoofolo RNA IsolationKit (e nang le kholomo ea ho tlosa gDNA)

Lisele Kakaretso ea RNA IsolationKit (e nang le kholomo ea ho tlosa gDNA)

Lnc-RT HeroTM I(With gDNase)(Super Premix for first-strand cDNA synthesis from lncRNA)

Nako ea 'Nete PCR EasyTM-SYBR Green I

Amelemo ofTlhahiso ea RNA:

Kit e sebelisa theknoloji ea ho ntša RNA ea moloko oa boraro, ha ho hlokahale ho sebelisa DNase.

Metsotso ea 11 ho tloha ho 96, 24, 12, le 6-cell plate cultured cell, e phahameng ka katleho e ntšoa ea gDNA-mahala, bohloeki bo phahameng, boleng bo phahameng ba RNA eohle.

Melemo ea lncRNA reverse transcript:

Sistimi ea reverse transcription e etselitsoeng LncRNA ho tlosa tšilafalo ea DNA kapele.

Sistimi ea ho fetolela ka morao e na le botsitso bo phahameng ba mocheso 'me e ntse e na le ts'ebetso e ntle ea ho fetolela ho 50°C.

Lithempleite tse nang le litaba tse phahameng tsa GC le sebopeho se rarahaneng sa bobeli li ka khutlisoa ka katleho e phahameng.

Melemoof qPCR:

Qalo e chesang ea Foregene Taq Polymerase e na le bokhoni bo phahameng ba ho holisa, kutlo e phahameng ea amplification, le lintlha tse phahameng tsa amplification.

The optimized Real PCR Easy Mix e etsa hore SYBR Green I e be le kutloisiso e phahameng ea ho lemoha, 'me matla a eona a fluorescence ke makhetlo a 3-5 a lihlahisoa tse tšoanang, tse ka finyellang litlhoko tsa mefuta e fapaneng ea liteko tsa bongata ba fluorescence.