Paul.xu: Created page with "Reagents The FAE solution consists of 98% formamide and 10 mM EDTA, prepared by mixing high-quality formamide (e.g., Sigma F9037) and RNA-grade 0.5M EDTA, pH 8.0. The FAE solu..."

2018-07-03T11:45:22Z

Created page with "Reagents The FAE solution consists of 98% formamide and 10 mM EDTA, prepared by mixing high-quality formamide (e.g., Sigma F9037) and RNA-grade 0.5M EDTA, pH 8.0. The FAE solu..."

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Reagents The FAE solution consists of 98% formamide and 10 mM EDTA, prepared by mixing high-quality formamide (e.g., Sigma F9037) and RNA-grade 0.5M EDTA, pH 8.0. The FAE solution can be prepared in advance and stored in aliquots at −80°C or −20°C for several months.

Harvesting cells BY4741 cells were grown in liquid YPD or synthetic media at 30°C. Before collecting cells, OD600 of cultures was measured with an Eppendorf Biophotometer. OD600 units of cell biomass, used to determine the amount of the FAE solution during cell lysis, were defined as OD600 × volume in ml (for example, precipitating 0.5 ml of culture with OD600 = 2.0 makes 1 OD600 unit). Cells were harvested by centrifugation for 30 s at 15,000 rpm in 1.5-ml microcentrifuge tubes using a tabletop microcentrifuge. After the cells were pelleted, medium was removed by pipetting or aspiration. Attention should be paid at this step to ensure complete removal of medium before FAE addition. This can be done by quick recentrifugation of the pelleted cells and careful aspiration of any residual medium from the top of the pellet. For larger culture volumes (5–25 ml), we pelleted cells first in 50-ml centrifuge tubes, resuspended pellets in 1 ml water, transferred the cells into 1.5-ml microfuge tubes and centrifuged them twice to remove liquid completely as described above. An additional wash of cell pellets with water may also be needed in experiments that involve treatments with high-salt media or any substances that can alter electrophoretic mobility of RNA if samples are loaded directly on gels without purification.

Using frozen cells Cell pellets can be frozen and kept at −80°C until lysis. To minimize RNA degradation, freezing should be done quickly (e.g., in racks prechilled at −80°C, using alcohol-dry ice bath, or in liquid nitrogen). For the same reason, frozen cells should not be allowed to thaw before being mixed with FAE.

FAE extraction of RNA Cell pellets in 1.5-ml microcentrifuge tubes prepared as described above (fresh or frozen) are resuspended in FAE by pipetting or brief vortexing. For optimal results, a minimum of 30–40 μl FAE per 1.0 OD600 unit of cell culture is recommended. The cell suspension in FAE is heated at 70°C for 10 min to extract RNA, briefly vortexed, and centrifuged at 12,500–15,000 rpm (14,000–21,000 × g) in a tabletop microcentrifuge at room temperature for 1–2 min. The FAE supernatant containing RNA is then transferred to a fresh tube. To avoid carrying over cells, we typically leave 5–10 μl supernatant on top of the cell pellet. The RNA in FAE can be used directly for gel analysis or optionally purified if desired. RNA in FAE can be stored at −20°C or −80°C for at least several months.

Purification of RNA from FAE extracts RNA can be precipitated from FAE extracts by first diluting them 5–10-fold with RNase-free water, followed by optional phenol-chloroform extraction and then adding 1/10 volume 3M sodium acetate (pH 5) and one volume of isopropanol. To obtain high-purity RNA using RNAzol RT (Molecular Research Center, Inc.), we mixed FAE extracts with RNAzol RT in a 1:3 to 1:7 ratio, added 0.4 volume of water relative to the combined volume of FAE and RNAzol RT and followed the manufacturer's protocol thereafter. The adjustment in the amount of water compared with the original protocol was necessary to ensure proper phase separation when the RNAzol reagent is mixed directly with formamide.

RT-PCR using FAE-extracted RNA A total of 1.5 μg of RNA was treated with 2 units of DNase I (Thermo Fisher) for 15 min at 37°C in a 100 μl reaction mixture supplemented with the manufacturer's buffer. The final formamide concentration in this reaction coming from FAE should not exceed 5%. RNA was purified by phenol/chloroform extraction and precipitated with isopropanol. RNA was next annealed with 0.4 μM oligo(dT)18 at 65°C for 5 min and reverse transcribed using the RevertAid enzyme (Thermo Fisher) for 1.5 h at 42°C. Reaction products were diluted with water and 1/30 was amplified by PCR with the SYBR FAST Universal qPCR Master Mix (Kapa Biosystems). All samples were analyzed in triplicate, using ACT1 as an internal control.
[[Category:Protocols]]

Extraction of RNA from Yeast - Revision history

Paul.xu: Created page with "Reagents The FAE solution consists of 98% formamide and 10 mM EDTA, prepared by mixing high-quality formamide (e.g., Sigma F9037) and RNA-grade 0.5M EDTA, pH 8.0. The FAE solu..."