Comparison of Different Fixation Methods for Combined Histological and Biomolecular Analysis of Fixed and Decalcified Bone Samples
The combination of histological and biomolecular analyses provides deep understanding of different biological processes and is of high interest for basic and applied research. However, the available analytical methods are still limited, especially when considering bone samples. This study compared d...
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| Format: | Article |
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MDPI AG
2022-07-01
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| Series: | Methods and Protocols |
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| Online Access: | https://www.mdpi.com/2409-9279/5/4/64 |
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| author | Sarah Al-Maawi Priscilia Valenzuela Eva Dohle Anja Heselich Robert Sader Shahram Ghanaati |
| author_facet | Sarah Al-Maawi Priscilia Valenzuela Eva Dohle Anja Heselich Robert Sader Shahram Ghanaati |
| author_sort | Sarah Al-Maawi |
| collection | DOAJ |
| description | The combination of histological and biomolecular analyses provides deep understanding of different biological processes and is of high interest for basic and applied research. However, the available analytical methods are still limited, especially when considering bone samples. This study compared different fixation media to identify a sufficient analytical method for the combination of histological, immuno-histological and biomolecular analyses of the same fixed, processed and paraffin embedded bone sample. Bone core biopsies of rats’ femurs were fixed in different media (RNAlater + formaldehyde (R + FFPE), methacarn (MFPE) or formaldehyde (FFPE)) for 1 week prior to decalcification by EDTA and further histological processing and paraffin embedding. Snap freezing (unfixed frozen tissue, UFT) and incubation in RNAlater were used as additional controls. After gaining the paraffin sections for histological and immunohistological analysis, the samples were deparaffined and RNA was isolated by a modified TRIZOL protocol. Subsequently, gene expression was evaluated using RT-qPCR. Comparable histo-morphological and immuno-histological results were evident in all paraffin embedded samples of MFPE, FFPE and R + FFPE. The isolated RNA in the group of MFPE showed a high concentration and high purity, which was comparable to the UFT and RNAlater groups. However, in the groups of FFPE and R + FFPE, the RNA quality and quantity were statistically significantly lower when compared to MFPE, UFT and RNAlater. RT-qPCR results showed a comparable outcome in the group of MFPE and UFT, whereas the groups of FFPE and R + FFPE did not result in a correctly amplified gene product. Sample fixation by means of methacarn is of high interest for clinical samples to allow a combination of histological, immunohistological and biomolecular analysis. The implementation of such evaluation method in clinical research may allow a deeper understanding of the processes of bone formation and regeneration. |
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| issn | 2409-9279 |
| language | English |
| last_indexed | 2024-03-09T04:01:56Z |
| publishDate | 2022-07-01 |
| publisher | MDPI AG |
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| series | Methods and Protocols |
| spelling | doaj.art-ded94750f0f142eba00b1106ab3224512023-12-03T14:12:33ZengMDPI AGMethods and Protocols2409-92792022-07-01546410.3390/mps5040064Comparison of Different Fixation Methods for Combined Histological and Biomolecular Analysis of Fixed and Decalcified Bone SamplesSarah Al-Maawi0Priscilia Valenzuela1Eva Dohle2Anja Heselich3Robert Sader4Shahram Ghanaati5Frankfurt Oral Regenerative Medicine (FORM-Lab), Clinic for Maxillofacial and Plastic Surgery, Goethe University, 60590 Frankfurt am Main, GermanyFrankfurt Oral Regenerative Medicine (FORM-Lab), Clinic for Maxillofacial and Plastic Surgery, Goethe University, 60590 Frankfurt am Main, GermanyFrankfurt Oral Regenerative Medicine (FORM-Lab), Clinic for Maxillofacial and Plastic Surgery, Goethe University, 60590 Frankfurt am Main, GermanyFrankfurt Oral Regenerative Medicine (FORM-Lab), Clinic for Maxillofacial and Plastic Surgery, Goethe University, 60590 Frankfurt am Main, GermanyFrankfurt Oral Regenerative Medicine (FORM-Lab), Clinic for Maxillofacial and Plastic Surgery, Goethe University, 60590 Frankfurt am Main, GermanyFrankfurt Oral Regenerative Medicine (FORM-Lab), Clinic for Maxillofacial and Plastic Surgery, Goethe University, 60590 Frankfurt am Main, GermanyThe combination of histological and biomolecular analyses provides deep understanding of different biological processes and is of high interest for basic and applied research. However, the available analytical methods are still limited, especially when considering bone samples. This study compared different fixation media to identify a sufficient analytical method for the combination of histological, immuno-histological and biomolecular analyses of the same fixed, processed and paraffin embedded bone sample. Bone core biopsies of rats’ femurs were fixed in different media (RNAlater + formaldehyde (R + FFPE), methacarn (MFPE) or formaldehyde (FFPE)) for 1 week prior to decalcification by EDTA and further histological processing and paraffin embedding. Snap freezing (unfixed frozen tissue, UFT) and incubation in RNAlater were used as additional controls. After gaining the paraffin sections for histological and immunohistological analysis, the samples were deparaffined and RNA was isolated by a modified TRIZOL protocol. Subsequently, gene expression was evaluated using RT-qPCR. Comparable histo-morphological and immuno-histological results were evident in all paraffin embedded samples of MFPE, FFPE and R + FFPE. The isolated RNA in the group of MFPE showed a high concentration and high purity, which was comparable to the UFT and RNAlater groups. However, in the groups of FFPE and R + FFPE, the RNA quality and quantity were statistically significantly lower when compared to MFPE, UFT and RNAlater. RT-qPCR results showed a comparable outcome in the group of MFPE and UFT, whereas the groups of FFPE and R + FFPE did not result in a correctly amplified gene product. Sample fixation by means of methacarn is of high interest for clinical samples to allow a combination of histological, immunohistological and biomolecular analysis. The implementation of such evaluation method in clinical research may allow a deeper understanding of the processes of bone formation and regeneration.https://www.mdpi.com/2409-9279/5/4/64biomolecular analysisRNAhistologymethacarnFFPEbone tissue |
| spellingShingle | Sarah Al-Maawi Priscilia Valenzuela Eva Dohle Anja Heselich Robert Sader Shahram Ghanaati Comparison of Different Fixation Methods for Combined Histological and Biomolecular Analysis of Fixed and Decalcified Bone Samples Methods and Protocols biomolecular analysis RNA histology methacarn FFPE bone tissue |
| title | Comparison of Different Fixation Methods for Combined Histological and Biomolecular Analysis of Fixed and Decalcified Bone Samples |
| title_full | Comparison of Different Fixation Methods for Combined Histological and Biomolecular Analysis of Fixed and Decalcified Bone Samples |
| title_fullStr | Comparison of Different Fixation Methods for Combined Histological and Biomolecular Analysis of Fixed and Decalcified Bone Samples |
| title_full_unstemmed | Comparison of Different Fixation Methods for Combined Histological and Biomolecular Analysis of Fixed and Decalcified Bone Samples |
| title_short | Comparison of Different Fixation Methods for Combined Histological and Biomolecular Analysis of Fixed and Decalcified Bone Samples |
| title_sort | comparison of different fixation methods for combined histological and biomolecular analysis of fixed and decalcified bone samples |
| topic | biomolecular analysis RNA histology methacarn FFPE bone tissue |
| url | https://www.mdpi.com/2409-9279/5/4/64 |
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