S. cerevisiae total RNA preparation and RT + quantitative PCR

GIM - Alain Jacquier's laboratory (Cosmin Saveanu)

Yeast total RNA preparation

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(protocol using hot phenol and no glass beads, adapted from Current Protocols in Molecular Biology)

Breaking the cells

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

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:
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):
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:

Quantitative PCR

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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On 20 May 2008, 18:03.