Both fresh-frozen and formalin-fixed,paraffinembedded（FFPE）human brain tissues are invaluable resources for molecular genetic studies of central nervous system diseases,especially neurodegenerative disorders.To iden...Both fresh-frozen and formalin-fixed,paraffinembedded（FFPE）human brain tissues are invaluable resources for molecular genetic studies of central nervous system diseases,especially neurodegenerative disorders.To identify the optimal method for DNA extraction from human brain tissue,we compared methods on differently-processed tissues.Fragments of LRRK2 and MAPT（257 bp and 483 bp/245 bp）were amplified for evaluation.We found that for FFPE samples,the success rate of DNA extraction was greater when using a commercial kit than a laboratory-based method（successful DNA extraction from 76%versus 33%of samples）.PCR amplicon size and storage period were key factors influencing the success rate of DNA extraction from FFPE samples.In the fresh-frozen samples,the DNA extraction success rate was 100%using either a commercial kit（QIAamp DNA Micro）or a laboratorybased method（sample boiling in 0.1 mol/L NaOH,followed by proteinase K digestion,and then DNA extraction using Chelex-100）regardless of PCR amplicon length or tissue storage time.Although the present results demonstrate that PCR-amplifiable genomic DNA can be extracted from both fresh-frozen and FFPE samples,fresh brain tissue is recommended for DNA extraction in future neuropathological studies.展开更多
The detection of single amino-acid variants （SAVs） usually depends on single-nucleotide polymorphisms （SNPs） database. Here, we describe a novel method that discovers SAVs at proteome level independent of SNPs dat...The detection of single amino-acid variants （SAVs） usually depends on single-nucleotide polymorphisms （SNPs） database. Here, we describe a novel method that discovers SAVs at proteome level independent of SNPs data. Using mass spectrometry-based de novo sequencing algorithm, peptide-candidates are identified and compared with theoretical protein database to generate SAVs under pairing strategy, which is followed by database re-searching to control false discovery rate. in human brain tissues, we can confidently identify known and novel protein variants with diverse origins. Combined with DNA/RNA sequencing, we verify SAVs derived from DNA mutations, RNA alternative splicing, and unknown post-transcriptional mechanisms. Furthermore, quantitative analysis in human brain tissues reveals several tissue-specific differential expressions of SAVs. This approach provides a novel access to high-throughput detection of protein variants, which may offer the potential for clinical biomarker discovery and mechanistic research.展开更多
基金supported by a Personnel Training Award from the Department of Health, Hebei Province, a Goldstar Award from the University of New South Wales, and an NHMRC Senior Principal Research Fellowship (630434)
文摘Both fresh-frozen and formalin-fixed,paraffinembedded（FFPE）human brain tissues are invaluable resources for molecular genetic studies of central nervous system diseases,especially neurodegenerative disorders.To identify the optimal method for DNA extraction from human brain tissue,we compared methods on differently-processed tissues.Fragments of LRRK2 and MAPT（257 bp and 483 bp/245 bp）were amplified for evaluation.We found that for FFPE samples,the success rate of DNA extraction was greater when using a commercial kit than a laboratory-based method（successful DNA extraction from 76%versus 33%of samples）.PCR amplicon size and storage period were key factors influencing the success rate of DNA extraction from FFPE samples.In the fresh-frozen samples,the DNA extraction success rate was 100%using either a commercial kit（QIAamp DNA Micro）or a laboratorybased method（sample boiling in 0.1 mol/L NaOH,followed by proteinase K digestion,and then DNA extraction using Chelex-100）regardless of PCR amplicon length or tissue storage time.Although the present results demonstrate that PCR-amplifiable genomic DNA can be extracted from both fresh-frozen and FFPE samples,fresh brain tissue is recommended for DNA extraction in future neuropathological studies.
文摘The detection of single amino-acid variants （SAVs） usually depends on single-nucleotide polymorphisms （SNPs） database. Here, we describe a novel method that discovers SAVs at proteome level independent of SNPs data. Using mass spectrometry-based de novo sequencing algorithm, peptide-candidates are identified and compared with theoretical protein database to generate SAVs under pairing strategy, which is followed by database re-searching to control false discovery rate. in human brain tissues, we can confidently identify known and novel protein variants with diverse origins. Combined with DNA/RNA sequencing, we verify SAVs derived from DNA mutations, RNA alternative splicing, and unknown post-transcriptional mechanisms. Furthermore, quantitative analysis in human brain tissues reveals several tissue-specific differential expressions of SAVs. This approach provides a novel access to high-throughput detection of protein variants, which may offer the potential for clinical biomarker discovery and mechanistic research.
