Chiyambi:

RNA, dzina la sayansi ribonucleic acid, chidule chake chimachokera ku RiboNucleic Acid, ndi mtundu wa asidi nucleic wopangidwa ndi kulumikiza osachepera angapo ribonucleotides kudzera phosphodiester zomangira.

Cholinga cha kufufuza RNA ndikuchotsa Total RNA yopanda kuipitsa, ndipo mfundo yofunikira ya kuchotsa RNA ikhoza kufotokozedwa mwachidule motere:

1. Kusokoneza ma cell kaye

2. Kenako chotsani zonyansa za macromolecular (mapuloteni, lipids, DNA, ndi zina).

3. Potsirizira pake, RNA imadziunjikira pamene ikuchotsa mchere ndi zosungunulira za organic, ndi kuyeretsanso RNA.

Zakale ndi zamakono za njira yochotsera

Mbadwo woyamba wa RNA m'zigawo njira ndi TRIzol mpweya njira (reagents ntchito monga guanidine isothiocyanate, phenol, chloroform, etc.) anatulukira ndi agogo aakazi dzina lake Piotr Chomczynski ndi agogo Nicoletta Sacchi, chifukwa chotulukira amalola RNA m'zigawo Quick ndi zosavuta, kuyambira RNA anayamba kufufuza.

Pambuyo pake, agogo anagulitsa chilolezo ichi ku Invitrogen, ndipo Invitrogen inalimbikitsa njira imeneyi padziko lonse lapansi ndipo inakhala njira yogwiritsidwa ntchito kwambiri.

Pambuyo pake, Invitrogen idagulidwa ndi Thermo!

Choyimira choyimira: Invitrogen's TRIzol reagent

Komabe, njira ya mpweya wa TRIzol imafuna mpweya wambiri ndi kuchotsa RNA, ndipo ma reagents omwe amagwiritsidwa ntchito ndi poizoni ndipo amatenga nthawi yaitali kuti agwire ntchito.

Chifukwa chake, njira yoyeretsera ya m'badwo wachiwiri wa enzyme idakhalapo, pogwiritsa ntchito nembanemba ya silika ya gel ngati chinthu china cha nucleic acid adsorption kukulitsa kuchira kwa RNA pachitsanzo ndikuchotsa zonyansa zina.

Njira ya m'badwo wachiwiri imapangitsa kuti ntchitoyi ikhale yosavuta chifukwa cha ntchito yokhazikika, imachepetsa chiopsezo, komanso imapangitsa kuti RNA ikhale yogwira ntchito bwino.

Zoyimira zoyimira: QIAGEN RNeasy ndi zinthu zambiri zapakhomo

Komabe, njira zonse za m'badwo woyamba ndi wachiwiri zimafuna kugwiritsa ntchito DNase kuchotsa kuipitsidwa kwa DNA, zomwe zimabweretsa zovuta zoyesa zotsatila zapansi.DNase yotsalira idzachepetsa mphamvu yolembera mobwereza ndikuchepetsa kaphatikizidwe ka cDNA ka chingwe choyamba, potero kukhudza kulondola kwa zotsatira.

Choncho, m'badwo wachitatu m'zigawo njira wokometsedwa pa maziko a m'badwo wachiwiri.Kuyesera sikufunikira kuwonjezera ma enzyme, ndipo RNA imatha kupezeka mosavuta ndi magawo awiri okha.

Mzere 1: DNA-Cleaning Column kuchotsa DNA

Mzere 2: Mzere wa RNA-okha umatsatsa ndikubwezeretsanso RNA

Ntchito yosavuta yotereyi sikuti imangopulumutsa nthawi, komanso imathandizira kugwira ntchito kwa zoyeserera zakutsika!

Zoyimira: QIAGEN RNeasy Plus ndiForegeneRNA purification series mankhwala

(RNeasy Plus)

(Foregene ndiPlant Total RNA Isolation Kit)

Ndikuuzeni mwakachetechete!Zida za RNA za Foregene zimaperekanso chofufutira cha RNase chosavuta kugwiritsa ntchito, chomwe chimatha kuchotseratu 400μg ya RNase pamalo olimba mu mphindi imodzi, osawonjezerapo chilichonse chapoizoni m'thupi la munthu!

Chithunzi1: Tchati chofananira cha ntchito

Zogulitsa za Foregene zitha kumaliza kutulutsa kwa RNA mu nyimbo ziwiri zokha, musapangitse ena kuchitira nsanje kupita kwanu patsogolo!

Chithunzi2:Ezotsatira za xperimental tchati chofananiza

M'badwo woyamba (1: TRIzol)

M'badwo wachiwiri (2: RNeasy of QIAGEN)

M'badwo wachitatu (3: QIAGEN's RNeasy kuphatikiza, 4: Foregene)

Chithunzi3: graph qPCR (makulidwe pamapindikira graph)

M'badwo woyamba (wobiriwira amatanthauza TRIzol -, wofiira amatanthauza TRIzol +)

M'badwo wachiwiri (buluu wakumwamba umatanthauza QIAGEN RNeasy-, wofiira amatanthauza QIAGEN RNeasy +)

M'badwo wachitatu (buluu amatanthauza QIAGEN RNeasy kuphatikiza, zobiriwira zikutanthauza Foregene)

(Zindikirani: qPCR graph "-" imasonyeza kuti DNase sinawonjezedwe ndipo kuipitsidwa kwa DNA sikunachotsedwe. Choncho, CT ndi yapamwamba ndipo zotsatira zake sizolondola; "+" imasonyeza kuti DNase yawonjezedwa ndipo kuipitsidwa kwa DNA kwachotsedwa, zotsatira zake ndi zolondola)

Kukweza kulikonse kwaukadaulo kumatanthauza kuti ntchito yoyeserera ikukula m'njira yosavuta komanso yosavuta kugwiritsa ntchito.

Foregene ikufuna kuthandiza ofufuza ambiri asayansi kuti asunge nthawi yoyesera ndi mtengo wake, kupeza zotsatira zoyeserera zapamwamba kwambiri, ndikuphatikizana kupanga zopambana zasayansi zambiri.