This HTML version may contain errors. Please use the pdf version when in doubt (protocol using hot phenol and no glass beads, adapted from Current Protocols in Molecular Biology)
Breaking the cells
Grow cells to mid-log phase (600 nm absorbance of 0.3-0.6, maximum 1)
Take an aliquot equivalent with 5 or 10 absorbance units (5-10
ODs) and recover the cells by centrifugation for 5 min. at 3500 rpm at
4 C.
Resuspend the pellet in 1 ml of sterile cooled
H2O
and transfer the suspension to a screw-capped tube
Spin down the cells by centrifugation (30 sec., 5000 rpm);
eliminate the supernatant. In this form, cells may be stored at -80 C
Heat at 65 C on a heated block for 30 min. with occasional vortexing
Place on ice 5 min. Centrifuge at top speed 5 min., 4 C
Recover supernatant and perform two additional phenol-chloroform extractions
TES (RNA buffer)
for 50 ml
10 mM Tris-HCl pH 7.5
0.5 ml of 1 M stock
10 mM EDTA
1 ml 0.5 M, pH8 stock
0.5% SDS
1.25 ml, 20% stock
Precipitating the RNA
Precipitate RNA with 1 ml of cold
Ethanol/NH4Acetate
(6 volumes/1 volume 7.5M) - 3.5 volumes to 1 volume RNA supernatant. Leave at 4 C for 10 min for efficient precipitation
Centrifuge at 4 C, maximum speed for 15 min
Wash the pellet with 800 ml Ethanol (70%) and recentrifuge
Eliminate the Ethanol, leave at air for a few minutes (until the pellet becomes transparent) and resuspend the pellet in 40 ml H2O
Measure the absorbance of a 1/200 dilution (1 unit of 260 nm absorbance corresponds to 40 mg/ml RNA). Adjust the concentration of the RNA solution to the smaller one (about 2.5 mg/ml)
Store at -20 C
DNAse cleaning of RNA
DNA copurifies with cellular RNA and should
be digested before any attempt of quantitative RT + PCR is done. A good
DNAse kit is sold by Ambion (TURBO DNA-free
kit). It contains both a recombinant form of bovine pancreatic DNAse I
and a resin that allows its removal at the end of the reaction. The
protocol used for 10mg total yeast RNA is
described below. In the absence of such a kit you can also use bovine
pancreatic DNAse I, purified to become RNAse-free. Low salt
concentration and the presence of Ca2+ ions are required for good activity:
Mix in a tube:
2 ml 10X buffer
0.4 ml DNAse (2 U)
x ml ARN solution
H2O qsp 15 ml
Incubate 40 min at 37 C
Add 2 ml
inactivation buffer
(beads suspension actually), vortex and leave for 2 min at room temperature
Centrifuge at 10 000 rpm, room temp, 1min 30 sec. Recover the supernatant.
Reverse transcription and quantitative PCR
Reverse transcription - RT
Reverse transcription of the RNA can be done either with random
oligonucleotides or with the reverse (3') oligonucleotide designed for
quantitative PCR. Two or more oligonucleotides, targeting different
RNAs may be used in a single RT reaction. In a first step, the
annealing of the oligonucleotides to the RNA is obtained in a manner
similar to the annealing step in a PCR reaction. In the second step,
elongation using a reverse transcriptase actually generates the cDNA
that will serve as a substrate for the quantitative PCR reaction. A
typical reaction will require about
1-2 mg
total RNA.
Annealing reaction (best in PCR tubes and in a PCR machine):
1 ml 5X RT buffer (250 mM Tris-HCI pH 8.3, 375 mM KCI, 15 mM MgCl2)1
0.5 ml oligonucleotide 1 (5 mM, 2.5 pmol)
0.5 ml oligonucleotide 2 (5 mM, 2.5 pmol)
... ml oligonucleotide x
x ml RNA solution qsp 5 ml
During annealing make up the mix containing the
reverse transcriptase. We use either SuperScript II or SuperScript III
from Invitrogen (formerly from Gibco). The mix will contain, for a
tube:
1 ml 5X RT buffer (250 mM Tris-HCI pH 8.3, 375 mM KCI, 15 mM MgCl2)
Once the RT product was obtained, quantitative PCR is done by usual protocols. The kit SYBRTM GREEN master mix from Applied Biosystem is a good choice. The first dilution used is obtained by mixing the 10 ml of RT reaction with 70 ml H2O. 5 ml of the dilution are mixed with 20 ml PCR mix that contains the two required oligonucleotides. Serial dilutions ensure linearity for the range measured.
ACT1 (in S. cerevisiae) is one of
the transcripts used for normalization. Other transcripts, like TAF9 or
TOM22, of different abundance may be chosen based on large scale
microarray experiments - those which do not vary under different growth
conditions are good candidates for consitutively expressed genes.
Footnotes:
1also called first strand buffer
2if the DNAse step is omitted, a good control is to do mock RT, without reverse transcriptase
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