Izici ze-lncRNA:

1. Ama-lncRNA avamise ukuba made, anenkulumo eguquguqukayo nezindlela ezihlukene zokuhlanganisa ngesikhathi sokuhlukaniswa;

2. Uma kuqhathaniswa nezakhi zofuzo, i-lncRNA ivamise ukuba ngaphansi;

3. Ama-lncRNA amaningi anencazelo esobala yesikhashana neyendawo enqubweni yokuhlukaniswa kwezicubu nokuthuthuka;

4. Kukhona ukubonakaliswa kwesici kumathumba nezinye izifo;

5. Izindawo ezingaphansi kwamaselula e-lncRNA zihlukahlukene;

6. Ukulandelana kuncane ekongiweni, kodwa umsebenzi unezinga elithile lokonga nokunye.

Isixazululo se-LncRNA sokuhlehla sokuloba:

Siyazi ukuthi i-lncRNA inesakhiwo sezinga eliphezulu kumaseli.I-RNA enjalo ivame ukuba nezakhiwo eziyinkimbinkimbi ezifana ne-stem loops noma izikhonkwane zezinwele.Uma isakhiwo sesibili se-lncRNA singakwazi ukuxazululwa ngempumelelo, ukuloba okuhlanekezelwe kuthambekele ekungaphumeleli noma kuphumelele lapho kwenziwa i-RT-qPCR.Inkinga yokusebenza kahle okuphansi.

Ngokuvamile, kunamaphuzu amathathu abalulekile ukuze sithole izifanekiso ze-cDNA zekhwalithi ephezulu:

Ikhwalithi yesifanekiso se-RNA, iziqalo zokuhlehla zokuloba kanye ne-reverse transcriptase, kanye ne-reverse transcriptase iyisici esiyinhloko esiyinhloko.I-RNA ehlanjululwe ngama-reagents e-RNA avame ukusetshenziswa ngokuvamile iqukethe izinsalela zotshwala kanye nosawoti we-guanidine, onemiphumela enamandla yokuvimbela kuma-transcriptases amaningi ahlanekezelwe;uma izinga lokushisa elilungile lokusabela kwe-reverse transcriptase liphansi, ngeke lilungele i-RNA.Ukuvulwa kwesakhiwo se-hierarchical.

Njengamanje, izinga lokushisa elifanele le-reverse transcriptase elisetshenziswa kumalabhorethri amaningi ngokuvamile lingu-42°C, futhi izinga lokushisa eliphezulu lingafinyelela cishe ku-50°C.Eqinisweni, ukuvulwa kwesakhiwo se-RNA esiphezulu kudinga izinga lokushisa eliphezulu.

Izinga lokushisa lokuhlanganiswa kwe-cDNA ye-strand yokuqala ye-Fortune Biotech 2×RT EasyTM Mix iphezulu njengo-50℃-65℃, okulungele ukuqaqa ngokuphelele isakhiwo sezinga eliphezulu se-lncRNA, futhi isikhathi sokusebenza sifushane.

Ngaphezu kwalokho, umkhiqizo futhi unamandla aqinile wokulwa nokuphazamiseka kwe-RNA enotshwala obuyinsalela nosawoti we-guanidine, ongahlangabezana ngokugcwele nezidingo zamalabhorethri ukuze kuzuzwe ukusebenza kahle kokubhala okuhlanekezelwe okuphezulu.

I-First Strand cDNA Synthesis Kit

Inombolo yomthetho: RT-01011 (izikhathi ezingama-25)

Izici:

1. Ukuzinza kwe-thermal: 65℃ isenomsebenzi omuhle wokuhlehla wokuloba, osiza ukuvula isakhiwo sesibili esiyinkimbinkimbi

2. Ukubekezelela: Ukuhlanganiswa kwe-cDNA kusengenziwa ngempumelelo kuzifanekiso ze-RNA ngokungahlanzeki kahle (izinsalela zikasawoti ze-ethanol ne-guanidine)

3. Ngokushesha: ukusebenza okulula, ukuhlanganiswa kokuqala kwe-cDNA ye-strand kungaqedwa kungakapheli imizuzu engama-25-35

Izinhlelo zokusebenza eziwela phansi: i-PCR, i-qPCR, i-RACE, ukulebula ama-chip, ukwakhiwa komtapo wolwazi, njll.

Ngaphezu kwalokho, i-FJ Biotech inikeza isethi egcwele yezixazululo ze-RT-PCR ukuhlangabezana nezidingo zezinhlobo ezahlukene zamakhasimende, okuhlanganisa nala makhithi alandelayo:

I-First Strand cDNA Synthesis Kit (Ye-qPCR)

Inombolo yomthetho: RT-01021 (izikhathi ezingama-50)

Izici:

1. Ukufaneleka: Iziqalo zokuhlehla zokulotshwa kwengezwe ukuze kwenziwe umsebenzi ube lula

2. Ukuqina kwe-thermal: 65℃ isenomsebenzi omuhle wokuhlehla wokulotshwa, osiza ukuvula isakhiwo sesibili esiyinkimbinkimbi

3. Ukubekezelela: Ukuhlanganiswa kwe-cDNA kusengenziwa ngempumelelo kuzifanekiso ze-RNA ngokungahlanzeki kahle

4. Ukuzwela: kulungele amasampula e-RNA ane-concentration ephansi

Uhlelo lokusebenza lwe-downstream: Ihlinzekelwe ngokukhethekile i-qPCR elandelayo

Ikhithi ye-RT-PCR enesinyathelo esisodwa

Inombolo yomthetho: RT-02011 (izikhathi eziyi-100)

Izici:

1. Ukuzinza kwe-thermal: 65℃ isenomsebenzi omuhle wokuhlehla wokuloba, osiza ukuvula isakhiwo sesibili esiyinkimbinkimbi

2. Ukubekezelela: Ukuhlanganiswa kwe-cDNA kusengenziwa ngempumelelo kuzifanekiso ze-RNA ngokungahlanzeki kahle

3. Ukucaciswa: I-PCR MIX ehlomile ithuthukisa ngempumelelo ukusebenza kahle kokukhulisa kanye nokucaciswa kokusabela

Izinhlelo zokusebenza eziphansi: i-cDNA cloning, ukuhlolwa kofuzo nokunye ukuhlola kwamangqamuzana

Ikhithi ye-RT-PCR enezinyathelo ezimbili

Inombolo yomthetho: RT-02021 (izikhathi eziyi-100)

Izici:

1. Kulula futhi kuyashesha: ukusabela kweshubhu elilodwa

2. Ukuqina kwe-thermal: 65℃ isenomsebenzi omuhle wokuhlehla wokulotshwa, osiza ukuvula isakhiwo sesibili esiyinkimbinkimbi

3. Ukucaciswa: I-Foregene Taq eyengeziwe ithuthukisa ngempumelelo ukusebenza kahle kokukhulisa kanye nokucaciswa kokusabela

Izinhlelo zokusebenza eziphansi: ukuhlolwa kwamangqamuzana njengokuhlolwa kofuzo

Isinyathelo esisodwa se-RT-qPCR kit-SYBR Green I

Inombolo yomthetho: RT-02111 (izikhathi eziyi-100)

Izici:

1. Okulula: ukusabela kweshubhu elilodwa

2. Ukuzwela okuphezulu: ingathola izifanekiso zamakhophi aphansi

3. Ukuvumelana: kulungele izinsimbi eziningi ze-fluorescence quantitative

Izinhlelo zokusebenza eziphansi: ukutholwa kwamasampula e-RNA afana namaseli namagciwane

Isinyathelo esisodwa se-RT-qPCR kit-Taqman

Inombolo yomthetho: RT-02131 (izikhathi eziyi-100)

Izici:

1. Ngokushesha: thola isakhi sofuzo esiqondiwe

2. Ukuzwela okuphezulu: ingathola izifanekiso zamakhophi aphansi

3. Ukuvumelana: kulungele izinsimbi eziningi ze-fluorescence quantitative

Izinhlelo zokusebenza eziphansi: ukutholwa kwamasampula e-RNA afana namaseli namagciwane
I-Forge Bioreverse Transcription Kit ingakusiza ukuxazulula kangcono inkinga yokuhlehla kokuloba.Ngesikhathi esifanayo, isebenzisana ne-Forge Bio RNA Extraction Kit (engakwazi ukususa ngempumelelo izinsalela ze-DNA) ukuze ikunikeze isisombululo esiphelele sokuhlola se-RNA.