Comparing protocols for preparation of DNA-free total yeast RNA suitable for RT-PCR.
BACKGROUND
Preparation of RNA free from DNA is a critical step before performing RT-PCR assay. Total RNA isolated from several sources, including those obtained from Saccharomyces cerevisiae, using routine methodologies are frequently contaminated with DNA, which can give rise to amplification products that mimic the amplicons expected from the RNA target.


RESULTS
We investigated the efficiency of two DNase I based protocols for eliminating DNA contaminations from RNA samples obtained from yeast cells. Both procedures are very efficient in eliminating DNA contamination from RNA samples and entail three main steps, which involve treating of RNA samples with DNase I, inhibition of the enzyme by EDTA and its subsequent inactivation at 65 degrees C. The DNase I treated samples were further purified with phenol: chloroform followed by precipitation with ice-cold ethanol (protocol I) or, alternatively, they were directly used in RT-PCR reactions (protocol II). Transcripts from ACT1, PDA1, CNA1, CNA2, TPS1 and TPS2 analyzed after each treatment showed that all mRNAs tested can be amplified if total RNA was extracted and purified after DNase I treatment, however, only TPS1, TPS2 and ACT1 mRNAs were amplified without extraction/purification step.


CONCLUSIONS
Although more laborious and requiring a higher initial amount of material, the inclusion of an extraction and purification step allows to prepare RNA samples that are free from DNA and from low molecular contaminants and can be applied to amplify any Saccharomyces cerevisiae mRNA by RT-PCR.
Del Aguila
Eduardo M
EM
Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Centro de Tecnologia, Bloco A, Lab. 545, CEP 21949-900, Rio de Janeiro (RJ), Brazil. eduardomere@hotmail.com
Dutra
Marcio B
MB
Silva
Joab T
JT
Paschoalin
Vânia M F
VM
D000199:Actins / Q000032:analysis; D017931:DNA Primers; D003850:Deoxyribonuclease I / Q000187:drug effects*; D004492:Edetic Acid; D004587:Electrophoresis, Agar Gel / Q000379:methods; D009696:Nucleic Acids / Q000378:metabolism*; D012331:RNA, Fungal / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis
C465210:Act1 protein, S cerevisiae; D000199:Actins; D017931:DNA Primers; D009696:Nucleic Acids; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D004492:Edetic Acid; D003850:Deoxyribonuclease I