Optimization of extraction and PCR amplification of RNA extracts from paraffin-embedded tissue in different fixatives.
A method was developed for fast and efficient isolation of RNA from paraffin-embedded tissue sections for subsequent PCR analysis. This method is based on the binding of RNA to acid-treated glass beads in the presence of a high molarity of guanidinium salt. It can be completed within an hour, and obviates the need for dewaxing and phenol/chloroform extractions. The effect of various fixatives and fixation times was tested and the amplification of actin mRNA fragments ranging in length from 82 to 507 bp was used to demonstrate the presence of RNA in the extracts. The method was compared to existing extraction techniques by studying the quality of the templates for reverse-transcriptase polymerase chain reaction amplification (RT-PCR), using virus-infected and mock-infected paraffin-embedded cell pellets as a model. PCR amplification of cellular and viral RNA was successful for RNA isolated by use of all extraction techniques, although the glass bead method was preferred for its simplicity and rapidity. Specimens fixed with formalin were found to be suitable for PCR, but the best results were obtained with acetone-fixed paraffin-embedded material. Dewaxing of tissue sections had no effect on the yield and quality of RNA extractions, and further purification of the extracts using gel filtration did not improve the results. After the protocols were optimized, rotavirus-infected cell pellets were used to demonstrate that extraction and amplification of dsRNA was possible. The information obtained from the studies with the model system was used for extraction of toroviral and rotaviral RNA from archival intestinal material. These data indicate that paraffin-embedded archival tissue can be used for RT-PCR analysis, adding an important technique to diagnostic pathology and retrospective studies.
Viral Gastroenteritis Section, Centers for Disease Control and Prevention, Atlanta, GA 30333.
M
M
Koopmans
S S
SS
Monroe
L M
LM
Coffield
S R
SR
Zaki
D000818:Animals; D001109:Archives; D001483:Base Sequence; D002417:Cattle; D002648:Child; D003967:Diarrhea / Q000382:microbiology; D006736:Horses; D006801:Humans; D007422:Intestines / Q000382:microbiology; D008969:Molecular Sequence Data; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D012330:RNA, Double-Stranded / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D014021:Tissue Preservation; D017003:Torovirus / Q000235:genetics / Q000302:isolation & purification*; D014777:Virus Diseases / Q000175:diagnosis*
D012330:RNA, Double-Stranded; D012367:RNA, Viral