Preparation of formaldehyde and pbs solution for long term tissue storage without damage of rna
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Formalin-fixed, paraffin-embedded tissues generally provide low yields of extractable RNA that exhibit both covalent modification of nucleic acid bases and strand cleavage. This frustrates efforts to perform retrospective analyses of gene expression using archival tissue specimens. A variety of conditions have been reported to demodify formaldehyde-fixed RNA in different model systems. We studied the reversal of formaldehyde fixation of RNA using a 50 base RNA oligonucleotide and total cellular RNA. Formaldehyde-adducted, native, and hydrolyzed RNA species were identified by their bioanalyzer electrophoretic migration patterns and RT–quantitative PCR. Demodification conditions included temperature, time, buffer, and pH. The reversal of formaldehyde-fixed RNA to native species without apparent RNA hydrolysis was most successfully performed in dilute Tris, phosphate, or similar buffers (pH 8) at 70°C for 30 minutes. Amines were not required for efficient formaldehyde demodification. Formaldehyde-fixed RNA was more labile than native RNA to treatment with heat and buffer, suggesting that antigen retrieval methods for proteins may impede RNA hybridization or RNA extraction. Taken together, the data indicate that reliable conditions may be used to remove formaldehyde adducts from RNA to improve the quality of RNA available for molecular studies.
Formaldehyde fixation followed by dehydration and paraffin embedding (FFPE) is commonly used to preserve tissue specimens for histological studies and provides most archival tissue samples. The isolation of high-quality RNA from FFPE tissues remains a challenge for molecular studies, despite the availability of multiple published and commercial methods.1 RNA degradation and formaldehyde modification of RNA appear to be the major contributors to this challenge. Degradation of RNA to low molecular weight species may be because of either sample treatment before and during fixation2 or long-term (1 year or longer) storage in paraffin.3 RNA extracted from FFPE tissues is usually fragmented to an average of 100 bases in length.4,5 Reproducible RT-PCR on FFPE-extracted RNA is limited to amplicons of fewer than 300 bases.6 Most laboratories strive to amplify segments of 150 or fewer bases. Degraded RNA can often be quantified by techniques that use short oligonucleotides, such as microarray and micro-RNA analyses, and by RT–quantitative PCR (qRT-PCR), but the results are almost invariably less sensitive and less reproducible than achieved using RNA extracted from fresh or fresh-frozen sources.7,8
Formaldehyde modification of nucleic acid bases reduces or blocks the base pairing necessary for molecular analysis by hybridization techniques. It is also responsible for cross-links to other macromolecules that reduce the yield of extracted RNA.9 An improved understanding of these modifications may lead to better strategies for their reversal and to the extraction of RNA that is more suitable for molecular analysis. Previous investigations demonstrated that formaldehyde-induced adducts, such as methylol (hydroxymethyl) groups and methylene bridge cross-links on the amine moiety of an adenine base, were reversible in model systems, such as mononucleotides9–11 and octamer ribonucleotides.12 Similar to the heat-induced antigen retrieval methods developed for the analysis of proteins,13 several groups3,12,14,15reported that heating the RNA extracted from FFPE tissues in dilute buffer improved the quality of RNA available for molecular studies, presumably by reversing formaldehyde modifications.
Although formaldehyde demodification treatment of FFPE-extracted RNA clearly improves molecular analyses, various studies report different results and demodification conditions. RNA extracted from FFPE tissues heated in Tris-EDTA yielded positive RT-PCR results for amplicons up to approximately 1700 bp.12 However, others16 were unable to reproduce these findings. Although some4,12used Tris buffers (pH 7 to 8) to remove formaldehyde adducts from RNA, others11,14 used citrate or Tris-acetate buffers (pH 4).
We initiated this study for three reasons. First, based on our knowledge of pH-dependent hydrolysis of RNA, we wanted to investigate whether optimized formaldehyde demodification conditions could be separated from those that produce hydrolysis. Second, we wanted to optimize conditions for the reversal of formaldehyde-induced RNA modifications. Third, we wanted to determine whether the ability of Tris buffer's amine to form Schiff bases and methylols was critical in its success as a demodification reagent. We reasoned that a 50 base RNA (50mer) oligonucleotide and total cellular RNA might produce more meaningful results than mononucleotides11 or RNA eightmers.12 The results of these experiments were expected to provide insights into the mechanism(s) of RNA degradation in FFPE tissues that could lead to improvements in the RNA obtained for molecular analyses.
Formaldehyde fixation followed by dehydration and paraffin embedding (FFPE) is commonly used to preserve tissue specimens for histological studies and provides most archival tissue samples. The isolation of high-quality RNA from FFPE tissues remains a challenge for molecular studies, despite the availability of multiple published and commercial methods.1 RNA degradation and formaldehyde modification of RNA appear to be the major contributors to this challenge. Degradation of RNA to low molecular weight species may be because of either sample treatment before and during fixation2 or long-term (1 year or longer) storage in paraffin.3 RNA extracted from FFPE tissues is usually fragmented to an average of 100 bases in length.4,5 Reproducible RT-PCR on FFPE-extracted RNA is limited to amplicons of fewer than 300 bases.6 Most laboratories strive to amplify segments of 150 or fewer bases. Degraded RNA can often be quantified by techniques that use short oligonucleotides, such as microarray and micro-RNA analyses, and by RT–quantitative PCR (qRT-PCR), but the results are almost invariably less sensitive and less reproducible than achieved using RNA extracted from fresh or fresh-frozen sources.7,8
Formaldehyde modification of nucleic acid bases reduces or blocks the base pairing necessary for molecular analysis by hybridization techniques. It is also responsible for cross-links to other macromolecules that reduce the yield of extracted RNA.9 An improved understanding of these modifications may lead to better strategies for their reversal and to the extraction of RNA that is more suitable for molecular analysis. Previous investigations demonstrated that formaldehyde-induced adducts, such as methylol (hydroxymethyl) groups and methylene bridge cross-links on the amine moiety of an adenine base, were reversible in model systems, such as mononucleotides9–11 and octamer ribonucleotides.12 Similar to the heat-induced antigen retrieval methods developed for the analysis of proteins,13 several groups3,12,14,15reported that heating the RNA extracted from FFPE tissues in dilute buffer improved the quality of RNA available for molecular studies, presumably by reversing formaldehyde modifications.
Although formaldehyde demodification treatment of FFPE-extracted RNA clearly improves molecular analyses, various studies report different results and demodification conditions. RNA extracted from FFPE tissues heated in Tris-EDTA yielded positive RT-PCR results for amplicons up to approximately 1700 bp.12 However, others16 were unable to reproduce these findings. Although some4,12used Tris buffers (pH 7 to 8) to remove formaldehyde adducts from RNA, others11,14 used citrate or Tris-acetate buffers (pH 4).
We initiated this study for three reasons. First, based on our knowledge of pH-dependent hydrolysis of RNA, we wanted to investigate whether optimized formaldehyde demodification conditions could be separated from those that produce hydrolysis. Second, we wanted to optimize conditions for the reversal of formaldehyde-induced RNA modifications. Third, we wanted to determine whether the ability of Tris buffer's amine to form Schiff bases and methylols was critical in its success as a demodification reagent. We reasoned that a 50 base RNA (50mer) oligonucleotide and total cellular RNA might produce more meaningful results than mononucleotides11 or RNA eightmers.12 The results of these experiments were expected to provide insights into the mechanism(s) of RNA degradation in FFPE tissues that could lead to improvements in the RNA obtained for molecular analyses.
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Formaldehyde fixation followed by dehydration and paraffin embedding (FFPE) is commonly used to preserve tissue specimens for histological studies and provides most archival tissue samples. The isolation of high-quality RNA from FFPE tissues remains a challenge for molecular studies, despite the availability of multiple published and commercial methods.1 RNA degradation and formaldehyde modification of RNA appear to be the major contributors to this challenge. Degradation of RNA to low molecular weight species may be because of either sample treatment before and during fixation2 or long-term (1 year or longer) storage in paraffin.3 RNA extracted from FFPE tissues is usually fragmented to an average of 100 bases in length.4,5 Reproducible RT-PCR on FFPE-extracted RNA is limited to amplicons of fewer than 300 bases.6 Most laboratories strive to amplify segments of 150 or fewer bases. Degraded RNA can often be quantified by techniques that use short oligonucleotides, such as microarray and micro-RNA analyses, and by RT–quantitative PCR (qRT-PCR), but the results are almost invariably less sensitive and less reproducible than achieved using RNA extracted from fresh or fresh-frozen sources.7,8
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