Effects of Fixation and Storage of Human Tissue Samples on Nucleic Acid Preservation
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Background: Because of recent advances in the molecular diagnosis of cancer patients, tissue quality has become more important in daily practice. Methods: To evaluate the effects of fixative, duration of fixation, decalcification, and storage periods on nucleic acid integrity, DNA and RNA were extracted from gastrointestinal cancer tissue. The yield and purity were analyzed, and polymerase chain reaction (PCR) for glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 60 bp), β-actin (148 bp), and human growth hormone (hGH; 434 bp) and real-time reverse transcription-PCR for β-actin (97 bp) were performed. Results: All formalin-fixed paraffin-embedded (FFPE) and methacarn-fixed paraffin-embedded (MFPE) samples tested positive for GAPDH and β-actin by PCR. hGH was successfully detected in all MFPE samples, but in only 46.7% of the FFPE samples. Prolonged formalin fixation resulted in fewer GAPDH and β-actin PCR products, andamplification of hGH was not successful. The PCR and reverse transcription-PCR results were significantly affected by the duration of decalcification. The yield, purity, and integrity of mRNA progressively decreased with increased storage periods of paraffin blocks. Conclusions: Fixation and storage should therefore be standardized in order to improve the quality of molecular pathologic diagnosis.
Abstract
Background: Because of recent advances in the molecular diagnosis of cancer patients, tissue quality has become more important in daily practice. Methods: To evaluate the effects of fixative, duration of fixation, decalcification, and storage periods on nucleic acid integrity, DNA and RNA were extracted from gastrointestinal cancer tissue. The yield and purity were analyzed, and polymerase chain reaction (PCR) for glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 60 bp), β-actin (148 bp), and human growth hormone (hGH; 434 bp) and real-time reverse transcription-PCR for β-actin (97 bp) were performed. Results: All formalin-fixed paraffin-embedded (FFPE) and methacarn-fixed paraffin-embedded (MFPE) samples tested positive for GAPDH and β-actin by PCR. hGH was successfully detected in all MFPE samples, but in only 46.7% of the FFPE samples. Prolonged formalin fixation resulted in fewer GAPDH and β-actin PCR products, andamplification of hGH was not successful. The PCR and reverse transcription-PCR results were significantly affected by the duration of decalcification. The yield, purity, and integrity of mRNA progressively decreased with increased storage periods of paraffin blocks. Conclusions: Fixation and storage should therefore be standardized in order to improve the quality of molecular pathologic diagnosis.
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