The Effect of Hollande's Fixative on the mRNA Levels in Normal Pancreas
DK Kaneshiro, Y Jiang, Y Wang, X Liu. Cleveland Clinic, Cleveland, OH; A Johnson & Johnson Company, San Diego, CA
Background: Buffered neutral formalin (BNF) is a widely used fixative in histology laboratories and is considered suitable for preservation of both DNA and RNA for molecular studies. Hollande's solution is also a commonly used fixative; however, its effects on mRNA levels have not been previously reported. This study aims to retrospectively determine mRNA levels for 3 endogenous genes [β-actin, hydroxymethylbilane synthase (HMBS), ribosomal protein L13 A (RPL13A)] and 2 additional genes [human equilibrium nucleotide transporter 1 (hENT1) and ribonucleotide reductase subunit 1 (RRM1)] in normal pancreas fixed in either Hollande's solution or BNF.
Design: Normal pancreas from 12 cases from years 2000-01 fixed using Hollande's solution, 9 cases from 2002-03 fixed using BNF, and 34 cases from 2004-05 fixed using BNF were included in the study. Unstained slides were microdissected to only include normal pancreas for RNA isolation. 100 ng total of RNA from each specimen was used for the one-step RTQ-PCR using paired primers for β-actin, HMBS, RPL13A, hENT1 and RRM1. The mRNA level was expressed by the cycle threshold (Ct) value. The results of the Hollande's fixed, BNF-fixed (2002-03) and BNF-fixed (2004-05) groups were compared using a Mann-Whitney U test, with a level of significance set to p<0.05. The mRNA level difference between groups was expressed for each gene by ΔCt and the fold (%) calculated using the formula (1/2ΔCt) x 100%.
Results: See Table 1 for results.
|Hollande's (N=12, 2000-01)||23.82||24.84||31.33||29.22||30.90|
|BNF (N=9, 2002-03)||21.89||22.27||29.68||25.99||28.26|
|BNF (N=34, 2004-05)||21.65||22.38||29.72||26.42||28.46|
|P value Hollande's vs BNF (2002-03)||0.0018||0.0072||0.0092||0.0117||0.0014|
|P value Hollande's vs BNF (2004-05)||0.0016||0.0013||0.0022||0.0059||0.0015|
|P value BNF (2002-03) vs BNF (2004-05)||0.368||0.243||0.323||0.965||0.693|
|Fold (%) [Hollande's/BNF (2002-03)]||14.53||7.64||19.21||12.06||13.85|
|Fold (%) [Hollande's/BNF (2004-05)]||11.38||8.57||19.84||18.44||16.55|