Sensitivity of the polymerase chain reaction for detecting human T-cell leukemia virus type I sequences in paraffin-embedded tissue. Effect of unbuffered formalin fixation.
Recently, the application of the polymerase chain reaction (PCR) to formalin-fixed paraffin-embedded tissue has been reported. But formalin, especially unbuffered formalin, is known to break DNA into small fragments. DNA extracted from MT-2 cells fixed in unbuffered formalin for various periods of time were subjected to the PCR and the effect of unbuffered formalin fixation on the ability of the PCR to detect exogenous sequences; i.e., human T-cell leukemia virus type I (HTLV-I) proviral DNA, was examined. The sensitivity of the PCR decreased as a function of both the duration of fixation and the length of the expected DNA products. When the expected length of the PCR product was about 200 bp, a slight decrease in the sensitivity was observed after 4-day fixation. When it was about 300 bp, a similar decrease was observed following 4-h fixation. In the case of a 500 bp product, the sensitivity began to decrease after 30-min fixation and a 100-fold decrease was observed after 10-day fixation. A decrease was not observed, however, with a 100 bp product. The appropriate design of primers, especially with regard to the length of the amplified product, is essential to keep the sensitivity of the PCR, particularly when the target tissues have been fixed in unbuffered formalin.
Ohara
Y
Y
Department of Neurological Sciences, Tohoku University School of Medicine, Sendai, Japan.
Honma
M
M
Iwasaki
Y
Y
D001483:Base Sequence; D002021:Buffers; D002478:Cells, Cultured; D004279:DNA, Viral / Q000032:analysis*; D005404:Fixatives / Q000494:pharmacology*; D005557:Formaldehyde / Q000494:pharmacology*; D015368:Human T-lymphotropic virus 1 / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D002021:Buffers; D004279:DNA, Viral; D005404:Fixatives; D005557:Formaldehyde