RT-PCR for the Detection of Translocations in Bone and Soft Tissue Tumours in Formalin-Fixed Paraffin-Embedded Tissues

2010 ◽  
pp. 257-268 ◽  
Author(s):  
Ann Williams ◽  
D. Chas Mangham
Keyword(s):  
Soft Tissue ◽  
Rt Pcr ◽  
Soft Tissue Tumours ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

scholarly journals Evaluation of the optimal provision of formalin-fixed, paraffin-embedded material for reverse transcription-PCR in soft-tissue tumour diagnosis

2016 ◽  
Vol 70 (1) ◽  
pp. 20-24 ◽  
Author(s):  
Khin Thway ◽  
Dorte Wren ◽  
Jasmin Lee ◽  
Lisa Thompson ◽  
Cyril Fisher ◽  
...  
Keyword(s):  
Soft Tissue ◽  
Reverse Transcription ◽  
Rna Extraction ◽  
Histopathological Diagnosis ◽  
Technical Failure ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

AimsMolecular genetic analysis is now a routine ancillary diagnostic modality to the histopathological diagnosis of soft-tissue neoplasms, many of which harbour characteristic gene fusions detectable by reverse transcription-PCR (RT-PCR). As the final diagnosis often depends on the molecular result, it is important to obtain the optimal yield of patient RNA.MethodsWe assessed the most reliable method of providing formalin-fixed, paraffin-embedded material for optimal RNA yield by comparing three consecutive periods in which different preparations (5×10 μm scrolls, 5×5 μm sections and 1×10 μm sections) were used for RNA extraction for RT-PCR, with its technical success rate.ResultsFor ‘2011’, ‘2012’ and ‘2013’, RT-PCR technical failure rates were 13.4%, 4.4% and 7.9%, respectively. The percentage of failed referral cases was 71.4%, 85.7% and 31.3%, and the proportion of core biopsy to excision specimens was 3:15, 2:5 and 13:3.ConclusionsThis study shows that the effectiveness of RNA extraction and purification is dependent on both specimen type and the tissue sectioning strategy. The failure rate has improved over recent years, particularly for large specimens as large numbers of thick 10 μm scrolls can saturate RNA extraction columns. In contrast, recent technical fails are more frequent in core biopsies, where 1×10 μm sections are insufficient for adequate RNA extraction. While previous technical fails occurred mostly in referred cases, this appears no longer the case due to the better fixation and processing of specimens in external surgical pathology departments because of the widespread recognition of the importance of molecular diagnostics as an important part of the patient pathway.

scholarly journals Apolipoprotein D Expression in Primary Brain Tumors: Analysis by Quantitative RT-PCR in Formalin-fixed, Paraffin-embedded Tissue

2005 ◽  
Vol 53 (8) ◽  
pp. 963-969 ◽  
Author(s):  
Stephen B. Hunter ◽  
Vijay Varma ◽  
Bahig Shehata ◽  
J.D.L. Nolen ◽  
Cynthia Cohen ◽  
...  
Keyword(s):  
Brain Tumors ◽  
Tumor Type ◽  
Rt Pcr ◽  
Who Grade ◽  
Primary Brain Tumors ◽  
Formalin Fixed Paraffin ◽  
Pilocytic Astrocytomas ◽  
Formalin Fixed Paraffin Embedded ◽  
Apolipoprotein D ◽  
Formalin Fixed

Apolipoprotein D (apoD) expression has been shown to correlate both with cell cycle arrest and with prognosis in several types of malignancy, including central nervous system astrocytomas and medulloblastomas. ApoD expression was investigated by real-time quantitative RT-PCR using RNA extracted from 68 formalin-fixed, paraffin-embedded brain specimens. Glyceraldehyde phosphate dehydrogenase was used as an internal control. Quantitation was achieved on all specimens. Sixteen poorly infiltrating WHO grade I glial neoplasms (i.e., pilocytic astrocytomas and gangliogliomas) showed an average 20-fold higher apoD expression level compared with the 20 diffusely infiltrating glial neoplasms (i.e., glioblastoma, anaplastic astrocytoma, oligodendrogliomas; p=0.00004). A small number of exceptions (i.e., two high-expressing glioblastomas and three low-expressing gangliogliomas) were identified. Analyzed as individual tumor groups, poorly infiltrating grade I pilocytic astrocytomas and gangliogliomas differed significantly from each tumor type within the diffusely infiltrating higher-grade category ( p<0.05 for each comparison) but not from each other ( p>0.05). Conversely, each individual tumor type within the diffusely infiltrating category differed significantly from both pilocytic astrocytomas and gangliogliomas ( p<0.05) but did not vary from other infiltrating tumors ( p>0.05). Ependymomas, non-infiltrating grade II neoplasms, expressed levels of apoD similar to or lower than levels expressed by the diffusely infiltrating gliomas. Ten medulloblastomas with survival longer than 3 years averaged slightly higher apoD expression than four fatal medulloblastomas; however, this result was not statistically significant and individual exceptions were notable. In 17 of the medulloblastomas, MIB-1 proliferation rates quantitated by image cytometry did not correlate with apoD expression. In addition, apoD expression was 5-fold higher in the slowly proliferating grade I glial neoplasms compared with non-proliferating normal brain tissue ( p=0.01), suggesting that apoD expression is not simply an inverse measure of proliferation. ApoD expression measured by quantitative RT-PCR may be useful in the differential diagnosis of primary brain tumors, particularly pilocytic astrocytomas and gangliogliomas.

Detection of West Nile virus in formalin-fixed, paraffin-embedded human tissues by RT-PCR: A useful adjunct to conventional tissue-based diagnostic methods

2007 ◽  
Vol 38 (2) ◽  
pp. 106-111 ◽  
Author(s):  
Julu Bhatnagar ◽  
Jeannette Guarner ◽  
Christopher D. Paddock ◽  
Wun-Ju Shieh ◽  
Robert S. Lanciotti ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Diagnostic Methods ◽  
Human Tissues ◽  
Rt Pcr ◽  
West Nile ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Sarcoma Subgrouping by Detection of Fusion Transcripts Using NanoString nCounter Technology

2018 ◽  
Vol 22 (3) ◽  
pp. 205-213 ◽  
Author(s):  
Javal Sheth ◽  
Anthony Arnoldo ◽  
Yunan Zhong ◽  
Paula Marrano ◽  
Carlos Pereira ◽  
...  
Keyword(s):  
Rt Pcr ◽  
Fusion Transcripts ◽  
Pediatric Sarcoma ◽  
Formalin Fixed Paraffin ◽  
Pediatric Sarcomas ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Nanostring Technology ◽  
Future Work ◽  
Formalin Fixed

Background NanoString technology is an innovative barcode-based system that requires less tissue than traditional techniques and can test for multiple fusion transcripts in a single reaction. The objective of this study was to determine the utility of NanoString technology in the detection of sarcoma-specific fusion transcripts in pediatric sarcomas. Design Probe pairs for the most common pediatric sarcoma fusion transcripts were designed for the assay. The NanoString assay was used to test 22 specific fusion transcripts in 45 sarcoma samples that had exhibited one of these fusion genes previously by reverse transcription polymerase chain reaction (RT-PCR). A mixture of frozen (n = 18), formalin-fixed, paraffin-embedded (FFPE) tissue (n = 23), and rapid extract template (n = 4) were used for testing. Results Each of the 22 transcripts tested was detected in at least one of the 45 tumor samples. The results of the NanoString assay were 100% concordant with the previous RT-PCR results for the tumor samples, and the technique was successful using both FFPE and rapid extract method. Conclusion Multiplexed interrogation for sarcoma-specific fusion transcripts using NanoString technology is a reliable approach for molecular diagnosis of pediatric sarcomas and works well with FFPE tissues. Future work will involve validating additional sarcoma fusion transcripts as well as determining the optimal workflow for diagnostic purposes.

scholarly journals Improved RT-PCR Amplification for Molecular Analyses with Long-term Preserved Formalin-fixed, Paraffin-embedded Tissue Specimens

2006 ◽  
Vol 54 (7) ◽  
pp. 773-780 ◽  
Author(s):  
Kiyohiro Hamatani ◽  
Hidetaka Eguchi ◽  
Keiko Takahashi ◽  
Kazuaki Koyama ◽  
Mayumi Mukai ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Rt Pcr ◽  
Molecular Analyses ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Tissue Specimens ◽  
Formalin Fixed

scholarly journals Targeted Resequencing RevealsALKFusions in Non-Small Cell Lung Carcinomas Detected by FISH, Immunohistochemistry, and Real-Time RT-PCR: A Comparison of Four Methods

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Katja Tuononen ◽  
Virinder Kaur Sarhadi ◽  
Aino Wirtanen ◽  
Mikko Rönty ◽  
Kaisa Salmenkivi ◽  
...  
Keyword(s):  
Real Time ◽  
Tumor Tissue ◽  
Small Cell ◽  
Rt Pcr ◽  
Small Cell Lung ◽  
Lung Carcinomas ◽  
Targeted Resequencing ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements occur in a subgroup of non-small cell lung carcinomas (NSCLCs). The identification of these rearrangements is important for guiding treatment decisions. The aim of our study was to screenALKgene fusions in NSCLCs and to compare the results detected by targeted resequencing with results detected by commonly used methods, including fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and real-time reverse transcription-PCR (RT-PCR). Furthermore, we aimed to ascertain the potential of targeted resequencing in detection ofALK-rearranged lung carcinomas. We assessedALKfusion status for 95 formalin-fixed paraffin-embedded tumor tissue specimens from 87 patients with NSCLC by FISH and real-time RT-PCR, for 57 specimens from 56 patients by targeted resequencing, and for 14 specimens from 14 patients by IHC. All methods were performed successfully on formalin-fixed paraffin-embedded tumor tissue material. We detectedALKfusion in 5.7% (5 out of 87) of patients examined. The results obtained from resequencing correlated significantly with those from FISH, real-time RT-PCR, and IHC. Targeted resequencing proved to be a promising method forALKgene fusion detection in NSCLC. Means to reduce the material and turnaround time required for analysis are, however, needed.

Fluorescence in situ analysis of soft tissue tumor associated genetic alterations in formalin-fixed paraffin-embedded tissue

2014 ◽  
Vol 210 (12) ◽  
pp. 804-811 ◽  
Author(s):  
Heike Horn ◽  
Jan Allmanritter ◽  
Claudio Doglioni ◽  
Alexander Marx ◽  
Justus Müller ◽  
...  
Keyword(s):  
Soft Tissue ◽  
Soft Tissue Tumor ◽  
Genetic Alterations ◽  
In Situ Analysis ◽  
Tissue Tumor ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

scholarly journals A rapid RT-PCR method for detection of intact RNA in formalin-fixed paraffin-embedded tissues

1995 ◽  
Vol 23 (15) ◽  
pp. 3071-3072 ◽  
Author(s):  
Yi-Hai Jiang ◽  
Laurie A. Davidson ◽  
Joanne R. Lupton ◽  
Robert S. Chapkin
Keyword(s):  
Rt Pcr ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Pcr Method ◽  
Formalin Fixed
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