For first-line non-small-cell lung cancer（NSCLC） therapy,detecting mutation status of the epidermal growth factor receptor（EGFR） gene constitutes a prudent test to identify patients who are most likely to benefit ...For first-line non-small-cell lung cancer（NSCLC） therapy,detecting mutation status of the epidermal growth factor receptor（EGFR） gene constitutes a prudent test to identify patients who are most likely to benefit from EGFR-tyrosine kinase inhibitor（TKI） therapy.Now,the material for detecting EGFR gene mutation status mainly comes from formalin-fixed and paraffin-embedded（FFPE） tissues.DNA extraction from FFPE and the amplification of EGFR gene by polymerase chain reaction（PCR） are two key steps for detecting EGFR gene mutation.We showed a simple method of DNA extraction from FFPE tissues for the effective amplification of EGFR gene.Extracting DNA from the FFPE tissues of NSCLC patients with 1% Triton X-100（pH=10.0） was performed by heating at 95 °C for 30 min.Meanwhile,a commercial kit was used to extract DNA from the same FFPE tissues of NSCLC patients for comparison.DNA extracted products were used as template for amplifying the exons 18,19,20 and 21 of EGFR by PCR for different amplified fragments.Results show that DNA fragment size extracted from FFPE tissues with 1% Triton X was about 250―500 base pairs（bp）.However,DNA fragment size extracted from FFPE tissues via commercial kit was about from several hundreds to several thousands bp.The DNA yield extracted from FFPE tissues with 1% Triton X was larger than that via commercial kit.For about 500 bp fragment,four exons of EGFR could not be amplified more efficiently from extracted DNA with 1% Triton X than with commercial kit.However,for about 200 bp fragment.This simple and non-laborious protocol could successfully be used to extract DNA from FFPE tissue for the amplification of EGFR gene by PCR,further screening of EGFR gene mutation and facilitating the molecular analysis of a large number of FFPE tissues from NSCLC patients.展开更多
Background: Differential diagnosis of follicular thyroid carcinoma (FTC) from follicular thyroid adenoma (FTA) is often difficult since presence or absence of capsular/vascular invasion can not be determined by preope...Background: Differential diagnosis of follicular thyroid carcinoma (FTC) from follicular thyroid adenoma (FTA) is often difficult since presence or absence of capsular/vascular invasion can not be determined by preoperative fine needle aspiration cytology, and may not be judged unanimously on permanent sections even among experienced pathologists. Determination of molecular-genetic factors such as trefoil factor 3 (TFF3) mRNA in the follicular thyroid tumors may be useful aid to improve the accuracy of diagnosis, though it is considered to be unstable and relatively low concentrated genetic substance. Purpose of our study is to investigate expression level of TFF3 mRNA of thyroid follicular tumors using formalin-fixed, paraffin-embedded (FFPE) tissue. Methods: Study population included FFPE sections from 19 FTC cases, 20 FTA cases, 11 adenomatous goiter (G) cases and 12 samples of normal thyroid tissue (N) adjacent to thyroid tumors. RNeasy FFPE kit was used for extraction of total RNA. Purification and concentration values were determined by spectrophotometer. Extracted RNA was used for cDNA synthesis in reverse transcription. Synthesized cDNA subsequently proceeded for relative quantification of TFF3 mRNA by RT-qPCR using TFF3 primers. Glyceroldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthin phosphorobosyltransferase1 (HPRT1) were used as control genes. The mean and standard deviation of TFF3 mRNA expression level were analyzed by software Multiplate RQ. Results: Extraction by the FFPE kit yielded high concentration of RNA in all cases. Purification values were 1.8 in average. Concentration values were significantly higher in FTC and FTA relative to G and N tissues, possibly due to high density of thyrocytes in the samples. Relative quantification of TFF3 mRNA expression level showed broad ranges both in FTC and FTA, while the analyses in G and N tissues indicated narrow ranges. Conclusion: FFPE tissues from thyroid follicular tumors can be used for measurement of unstable and low concentrated genetic substances such as TFF3 mRNA. Its diagnostic value yet remains to be determined.展开更多
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
基金Supported by the Jilin Science & Technology Development Plan,China(No.201201060)the Scientific Research Foundation of Jilin Province,China(No.20100942)+1 种基金the Fund of Developing and Reforming Community of Jilin Province,China(No.2010-1928)the Health Scientific Research Foundation of Jilin Province,China(Nos.2009z081,2010Z083)
文摘For first-line non-small-cell lung cancer（NSCLC） therapy,detecting mutation status of the epidermal growth factor receptor（EGFR） gene constitutes a prudent test to identify patients who are most likely to benefit from EGFR-tyrosine kinase inhibitor（TKI） therapy.Now,the material for detecting EGFR gene mutation status mainly comes from formalin-fixed and paraffin-embedded（FFPE） tissues.DNA extraction from FFPE and the amplification of EGFR gene by polymerase chain reaction（PCR） are two key steps for detecting EGFR gene mutation.We showed a simple method of DNA extraction from FFPE tissues for the effective amplification of EGFR gene.Extracting DNA from the FFPE tissues of NSCLC patients with 1% Triton X-100（pH=10.0） was performed by heating at 95 °C for 30 min.Meanwhile,a commercial kit was used to extract DNA from the same FFPE tissues of NSCLC patients for comparison.DNA extracted products were used as template for amplifying the exons 18,19,20 and 21 of EGFR by PCR for different amplified fragments.Results show that DNA fragment size extracted from FFPE tissues with 1% Triton X was about 250―500 base pairs（bp）.However,DNA fragment size extracted from FFPE tissues via commercial kit was about from several hundreds to several thousands bp.The DNA yield extracted from FFPE tissues with 1% Triton X was larger than that via commercial kit.For about 500 bp fragment,four exons of EGFR could not be amplified more efficiently from extracted DNA with 1% Triton X than with commercial kit.However,for about 200 bp fragment.This simple and non-laborious protocol could successfully be used to extract DNA from FFPE tissue for the amplification of EGFR gene by PCR,further screening of EGFR gene mutation and facilitating the molecular analysis of a large number of FFPE tissues from NSCLC patients.
文摘Background: Differential diagnosis of follicular thyroid carcinoma (FTC) from follicular thyroid adenoma (FTA) is often difficult since presence or absence of capsular/vascular invasion can not be determined by preoperative fine needle aspiration cytology, and may not be judged unanimously on permanent sections even among experienced pathologists. Determination of molecular-genetic factors such as trefoil factor 3 (TFF3) mRNA in the follicular thyroid tumors may be useful aid to improve the accuracy of diagnosis, though it is considered to be unstable and relatively low concentrated genetic substance. Purpose of our study is to investigate expression level of TFF3 mRNA of thyroid follicular tumors using formalin-fixed, paraffin-embedded (FFPE) tissue. Methods: Study population included FFPE sections from 19 FTC cases, 20 FTA cases, 11 adenomatous goiter (G) cases and 12 samples of normal thyroid tissue (N) adjacent to thyroid tumors. RNeasy FFPE kit was used for extraction of total RNA. Purification and concentration values were determined by spectrophotometer. Extracted RNA was used for cDNA synthesis in reverse transcription. Synthesized cDNA subsequently proceeded for relative quantification of TFF3 mRNA by RT-qPCR using TFF3 primers. Glyceroldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthin phosphorobosyltransferase1 (HPRT1) were used as control genes. The mean and standard deviation of TFF3 mRNA expression level were analyzed by software Multiplate RQ. Results: Extraction by the FFPE kit yielded high concentration of RNA in all cases. Purification values were 1.8 in average. Concentration values were significantly higher in FTC and FTA relative to G and N tissues, possibly due to high density of thyrocytes in the samples. Relative quantification of TFF3 mRNA expression level showed broad ranges both in FTC and FTA, while the analyses in G and N tissues indicated narrow ranges. Conclusion: FFPE tissues from thyroid follicular tumors can be used for measurement of unstable and low concentrated genetic substances such as TFF3 mRNA. Its diagnostic value yet remains to be determined.
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.
