En effektiv metode til isolering af stærkt Renset RNA fra Frø til brug i Kvantitative Transkriptomet Analysis
Summary
We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.
Abstract
Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.
Introduction
Planter producerer frø, som giver anledning til den næste generation. Frø akkumulere store mængder af opbevaring reserver, såsom olier, kulhydrater og proteiner, til post-germinative vækst. Mennesker anvender frø opbevaring reserver som kilder til fødevarer og dyrefoder, og dermed plantefrø er en af de største leverandører af spiselige organisk stof i hele verden. Stigende frøudbytter er en vigtig udfordring i plante videnskab.
Da frø opbevaring reserver er kommercielt værdifulde fødekilder og industrielle materialer, har de molekylære mekanismer, der ligger til grund for reguleringen af metabolismen af disse reserver blevet bredt undersøgt 1-6. Yderligere belyse disse mekanismer vil være nyttige for at øge frøudbytter i afgrøder. Frø udvikle sig i plante æggestokke efter befrugtningen, og de modnes gennem en række udviklingsstadier 1,6,7. Yderligere forstå den molekylære mekanisme underliggende frø udvikling kræver detaljeret, Præcise genekspressionsprofiler fra en række udviklende frø, der skal fremstilles. Imidlertid er de høje mængder af olier, proteiner, kulhydrater og polyphenoler i plantefrø gør det vanskeligt at isolere højt oprenset RNA, som udelukker nøjagtig profilering af genekspression.
Her introducerer vi en effektiv fremgangsmåde til RNA-isolering fra oliefrø indeholder store mængder af olier, proteiner og polyphenoler. Ved hjælp af denne metode, vil forskerne kunne udarbejde højt oprenset RNA. En sådan RNA vil være nyttig til overvågning transkriptionelle ændringer i vigtige gener, der kontrollerer den metaboliske regulering af frø opbevaring reserver i at udvikle og modne oliefrø.
Protocol
Representative Results
Discussion
Genekspressionsprofiler bidrage til vores forståelse af plantefysiologi; Derfor har specifikke RNA isolation metoder blevet udviklet til hver prøve tilstand 9-12. Vi undersøgte de processer, der blev inhiberet under RNA-isolering fra frø og fundet, at RNA-binding til silica-membraner blev alvorligt inhiberet. Store mængder olie, proteiner og polyfenoler hæmme RNA isolation. Vi ændret RNA-ekstraktion for at fjerne disse forbindelser med en lyseopløsning før processen af RNA-binding til silica me…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi takke personalet i Functional Genomics faciliteten og spektrografi og Bioimaging Facility, NIBB Core forskningsfaciliteter, og model Plant Research Facility, NIBB Bioressource Center.
Materials
| RNeasy Plant Mini Kit | QIAGEN | 74904 | |
| polyvinylpyrrolidone | Sigma-Aldrich | P5288-100G | |
| HOMOGENIZER S-303 | AS ONE | 1-1133-02 | |
| NanoDrop Lite | Thermo Scientific | ND-NDL-US-CAN | |
| PrimeScript RT reagent Kit (Perfect Real Time) | TAKARA | RR037A | |
| KAPA SYBR Fast qPCR kit | Kapa biosystems | KK4601 |
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