Effect of duration of fixation on quantitative XXXX polymerase chain reaction analyses.

@entity3334 @entity1697 @entity7 @entity5 @entity470 @entity957 @entity3544 @entity406 @entity982 @entity2543 @entity16757
Increasingly, there is the need to analyze gene expression in @entity5 tissues and correlate these findings with clinical outcome. Because there are few tissue banks containing enough frozen material suitable for large-scale genetic analyses, methods to isolate and quantify messenger RNA (mRNA) from @entity406 -fixed, @entity982 -embedded tissue sections are needed. Recovery of RNA from routinely processed biopsies and quantification by the polymerase chain reaction (PCR) has been reported; however, the effects of @entity406 fixation have not been well studied. We used a proteinase K-salt precipitation RNA isolation protocol followed by TaqMan quantitative PCR to compare the effect of @entity406 fixation for 24, 48, and 72 hours and for 1 week in normal (2), @entity2543 (3), and @entity957 (4) specimens yielding 9 fresh and 36 @entity406 -fixed samples. We also compared mRNA and protein expression levels using immunohistochemistry for @entity470 ( @entity470 ), @entity3334 , @entity3544 , and @entity7 ( @entity7 ) in 15 randomly selected and routinely processed @entity1697 . We were able to extract RNA suitable for quantitative @entity16757 ( @entity16757 ) from all fresh (9/9) and @entity406 -fixed (36/36) specimens fixed for differing lengths of time and from all (15/15) randomly selected @entity957 . We found that prolonged @entity406 fixation (>48 h) had a detrimental effect on quantitative @entity16757 polymerase chain reaction results that was most marked for @entity3334 and @entity7 but less evident for @entity3544 and @entity470 . Comparisons of quantitative @entity16757 polymerase chain reaction and immunohistochemistry showed that for all markers, except @entity3544 , there was good correlation between mRNA and protein levels. @entity3544 mRNA was overexpressed in only one case, but protein levels were elevated in all but one @entity5 , consistent with the established translational regulation of @entity3544 . These results show that RNA can be reliably isolated from @entity406 -fixed, @entity982 -embedded tissue sections and can produce reliable quantitative @entity16757 -PCR data. However, results for some markers are adversely affected by prolonged @entity406 fixation times.
