TY - JOUR
T1 - Non-formalin fixative versus formalin-fixed tissue
T2 - A comparison of histology and RNA quality
AU - Groelz, Daniel
AU - Sobin, Leslie
AU - Branton, Philip
AU - Compton, Carolyn
AU - Wyrich, Ralf
AU - Rainen, Lynne
N1 - Funding Information:
The authors wish to thank Nadine Dettmann, Isabell Blassnig and Evelyn Traenert for their technical assistance. The research leading to these results has received funding from the European Union Seventh Framework Programme [FP7/2007–2013] under grant agreement no. 222916 .
PY - 2013/2
Y1 - 2013/2
N2 - Preanalytical handling of tissue samples can influence bioanalyte quality and ultimately outcome of analytical results. The aim of this study was to compare RNA quality, performance in real time RT PCR and histology of formalin-fixed tissue to that of tissue fixed and stabilized with a formalin-free fixative, the PAXgene Tissue System (PAXgene), in an animal model under highly controlled preanalytical conditions. Samples of rat liver, kidney, spleen, intestine, lung, heart muscle, brain, and stomach tissue were either fixed in formalin or fixed in PAXgene or fresh frozen in liquid nitrogen. RNA was extracted from all samples, examined for integrity in microcapillary electrophoresis, and used in a series of quantitative RT PCR assays with increasing amplicon length. Histology of paraffin-embedded samples was determined by staining with hematoxylin and eosin.Histology of all formalin-fixed and PAXgene fixed samples was comparable. RNA with acceptable integrity scores could be isolated from all embedded tissues, 4.0 to 7.2 for formalin and 6.4 to 7.7 for PAXgene, as compared to 8.0 to 9.2 for fresh frozen samples. While RNA with acceptable RINs (RNA integrity number) could be isolated from formalin-fixed samples, in microcapillary electrophoresis this RNA separated with a slower migration rate and displayed diffuse, less focused peaks for ribosomal RNA as compared to RNA from frozen or PAXgene fixed samples. Furthermore, RNA from formalin-fixed tissues exhibited inhibition in quantitative RT PCR assays which increased with increasing amplicon length, while RNA from PAXgene fixed samples did not show such inhibition.In conclusion, our results demonstrate that excluding other preanalytical factors, PAXgene Tissue System preserves histology similarly to formalin, but unlike formalin, does not chemically modify RNA. RNA purified from PAXgene fixed tissues is of high integrity and performs as well as RNA from fresh frozen tissue in RT PCR regardless of amplicon length.
AB - Preanalytical handling of tissue samples can influence bioanalyte quality and ultimately outcome of analytical results. The aim of this study was to compare RNA quality, performance in real time RT PCR and histology of formalin-fixed tissue to that of tissue fixed and stabilized with a formalin-free fixative, the PAXgene Tissue System (PAXgene), in an animal model under highly controlled preanalytical conditions. Samples of rat liver, kidney, spleen, intestine, lung, heart muscle, brain, and stomach tissue were either fixed in formalin or fixed in PAXgene or fresh frozen in liquid nitrogen. RNA was extracted from all samples, examined for integrity in microcapillary electrophoresis, and used in a series of quantitative RT PCR assays with increasing amplicon length. Histology of paraffin-embedded samples was determined by staining with hematoxylin and eosin.Histology of all formalin-fixed and PAXgene fixed samples was comparable. RNA with acceptable integrity scores could be isolated from all embedded tissues, 4.0 to 7.2 for formalin and 6.4 to 7.7 for PAXgene, as compared to 8.0 to 9.2 for fresh frozen samples. While RNA with acceptable RINs (RNA integrity number) could be isolated from formalin-fixed samples, in microcapillary electrophoresis this RNA separated with a slower migration rate and displayed diffuse, less focused peaks for ribosomal RNA as compared to RNA from frozen or PAXgene fixed samples. Furthermore, RNA from formalin-fixed tissues exhibited inhibition in quantitative RT PCR assays which increased with increasing amplicon length, while RNA from PAXgene fixed samples did not show such inhibition.In conclusion, our results demonstrate that excluding other preanalytical factors, PAXgene Tissue System preserves histology similarly to formalin, but unlike formalin, does not chemically modify RNA. RNA purified from PAXgene fixed tissues is of high integrity and performs as well as RNA from fresh frozen tissue in RT PCR regardless of amplicon length.
KW - H&E histology
KW - PAXgene Tissue
KW - Quantitative RT PCR
KW - RNA
KW - RNA integrity score
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U2 - 10.1016/j.yexmp.2012.07.002
DO - 10.1016/j.yexmp.2012.07.002
M3 - Article
C2 - 22814231
AN - SCOPUS:84870914538
SN - 0014-4800
VL - 94
SP - 188
EP - 194
JO - Experimental and Molecular Pathology
JF - Experimental and Molecular Pathology
IS - 1
ER -