Protocol for gene expression analysis using Saccharomyces cerevisiae cells
Method Article
Gene expression analysis in yeast cells
https://doi.org/10.1038/protex.2019.009
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Protocol for gene expression analysis using Saccharomyces cerevisiae cells
-LETS buffer: 0.1 M LiCI, 0.01 M EDTA, 0.01 M Tris-HCI pH 7.4, 0.2% SDS
-Chloroform:Isoamylic Alcohol (24:1)
Lithium Chloride 5M
Ethanol 70%
NaAc 0.3M
TURBO DNase (AM2238, Ambion)
EDTA 150 mM
OligodT
Improm-II® Reverse Transcriptase (M314A, PROMEGA)
RNasin® Ribonuclease Inhibitor (N2118, PROMEGA)
SYBR® Premix Ex TaqTM Tli RNase H Plus Green with ROX (RR420A,Takara).
FastPrep Precellys24 (Bertin technologies)
NanoDrop 2000 Spectophotometer® (Thermo scientific).
DNA Engine Peltier Thermal Cycler (Bio Rad)
Sample collection
Collect approximately 2 × 108 cells from exponential growing yeast cultures
Pellet cells by centrifugation at 4000xg at 4ºC and wash once with cold RNase-free water
Cells can be stored at -20ºC
Lysis and RNA extraction
Resuspend cells with 500 μL of LETS buffer
Add one volume of saturated phenol (pH 4.5) and 200 μL of glass beads
Broke cells in a FastPrep Precellys24 (Bertin technologies)
Centrifuge cells at 13400xg for 5 min at 4°C
Transfer the aqueous phase to a new eppendorf
Add one volume of phenol:chloroform (5:1)
Centrifuge cells at 13400g for 10 min at 4°C
Repeat steps 8 to 10
Transfer the aqueous phase to a new eppendorf
Add one volume of chloroform:isoamylic alcohol (24:1)
Precipitate the RNA overnight with one volume of 5 M lithium chloride at -80°C.
Centrifuge cells at 13400xg for 15 min at 4°C
Wash the precipitate was with 70% ethanol
Centrifuge cells at 13400xg for 15 min at 4°C to collect the RNA
Resuspend RNA pellet with 200 μL of RNase-free water
Re-precipitate RNA at -80°C for 3h adding Na Acetate 0.3M and two volumes of ethanol
Centrifuge cells at 13400g for 15 min at 4°C
Wash the precipitate was with 70% ethanol
Centrifuge cells at 13400xg for 15 min at 4°C to collect the RNA
Resuspend RNA pellet in 30-50 μL of RNase-free water
RNA was quantified by measuring the absorbance at 260 nm with NanoDrop 2000 Spectophotometer® (Thermo scientific)
Load 1 μg of RNA in a 1% TAE agarose gel and run the electrophoresis 15 minutes at 120V to check the purified RNA and the sample integrity
cDNA synthesis
Incubate 5 μg of RNA with Turbo DNase (Ambion) for 1 hour at 37°C
Inactivate DNase by incubating the sample 10 minutes at 75°C in 15 mM EDTA
Incubate 1 μg of this RNA with 100 pmoles of oligo dT 5 minutes at 70°C
Transfer the sample to ice for 10 minutes
Incubated 5 min at 25ºC, 60 min at 42ºC and 5 min at 72ºC with Improm-II® Reverse Transcriptase and Recombinant RNasin® (Promega) following the manufacturer instructions
Transfer the sample to ice
The cDNA was analized by semiquantitative RT-PCR or by quantitative RT-PCR in a DNA Engine Peltier Termal Cycler (Bio Rad) using the SYBR® Premix Ex TaqTM Tli RNase H Plus Green with ROX (Takara)
The authors declare no competing financial interests
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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