A simple method for detection of rabies viral sequences in 16-year old archival brain specimens with one-week fixation in formalin.
Archival formalin-fixed and paraffin-embedded brain tissues are important source for diagnosis and molecular analysis. However, nucleic acids are particularly vulnerable to degradation during tissue processing. The brain cutting process usually is performed after 1 week of brain storage in formalin followed by embedding of each particular neuro-anatomical specimen in paraffin. A simple method of deparaffinization, proteinase K digestion and RNA extraction using the Boom technique to obtain rabies RNA in unbuffered, formalin-fixed and paraffin-embedded brain tissues kept at 30 degrees C for 16 years is described. Reverse transcription-polymerase chain reaction (RT-PCR) can be used to identify rabies viral N gene sequences of 150 bases in length in all patients, but not from every immunohistochemical (IHC)-positive specimen. Direct sequencing of 301bp of N gene was achieved in 4 of 7 patients. Results of sequencing a single sample of 1432 bases of N gene from a 24h processed formalin-fixed and paraffin-embedded rabies infected brain tissue after 1 month storage were in accord with those from frozen specimen analysis. It is strongly suggested that for further molecular analysis, a piece of fresh brain tissue should be saved prior to the brain sectioning process and stored no longer than 24h in formalin before embedding.
Wacharapluesadee
Supaporn
S
Molecular Biology Laboratory for Neurological Diseases, Department of Medicine, Chulalongkorn University Hospital, Bangkok, Thailand. spwa02@yahoo.com
Ruangvejvorachai
Preecha
P
Hemachudha
Thiravat
T
D000818:Animals; D001921:Brain / Q000821:virology*; D004285:Dogs; D005557:Formaldehyde; D006146:Guanidines; D006801:Humans; D019590:Nucleocapsid Proteins / Q000235:genetics; D010232:Paraffin; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D011818:Rabies / Q000175:diagnosis* / Q000821:virology; D011820:Rabies virus / Q000235:genetics / Q000302:isolation & purification*; D011933:Reagent Kits, Diagnostic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013861:Thiocyanates; D013997:Time Factors; D016707:Tissue Fixation / Q000379:methods*
D006146:Guanidines; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D013861:Thiocyanates; C099601:nucleocapsid protein, Rabies virus; D005557:Formaldehyde; C054436:guanidine thiocyanate; D010232:Paraffin