Long-term stability of RNA from FFPE brian tissue post-sectioning on slides.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e22142-e22142
Author(s):  
Donald James Witt ◽  
Steven M. Anderson ◽  
Briana King ◽  
Christina Borrego ◽  
Marcia Eisenberg ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Room Temperature ◽  
Storage Period ◽  
Ffpe Tissue ◽  
Formalin Fixation ◽  
Tissue Preparation ◽  
Rna Integrity ◽  
Paraffin Embedding ◽  
Mutational Status

e22142 Background: Analysis of nucleic acids (NA) from formalin fixed paraffin embedded (FFPE) tissue can provide detailed information about gene sequence mutational status, which may be important for oncology treatment decisions. FFPE specimens also have utility for retrospective analyses. Potential degradation of NA during formalin fixation, paraffin embedding processes and possible continued deterioration during subsequent storage may diminish utility of FFPE tissue for these purposes. Using real-time PCR, this study investigated the functional stability of RNA from brain FFPE tissue sections on slides over an extended time period after sectioning. Methods: Brain biopsy specimens obtained from glioblastoma patients with informed consent were used to prepare blocks with standard formalin fixation and paraffin embedding techniques. Slides were made from the FFPE blocks and stored at room temperature until testing. RNA was extracted from sequential slides within one week of sectioning for a zero time and then at 4, 8 and 12 months. Reverse transcription PCR was performed, and real-time PCR was analyzed on the ABI7900 to detect EGFRvIII mutation and cABL gene. RNA Integrity Analysis was performed with an Agilent Bioanalyzer. Results: Consistent qualitative results were obtained with EGFRvIII mutant positive specimens (n =10) and wild type (wt) specimens (n =10) from slides stored up to twelve months at room temperature compared to the initial testing (95% agreement). One wt specimen showed negative results for the first three time points but a low positive result at 12 months, possibly due to tumor content change in the different sections of the FFPE block. Ct values for EGFR (wt and mutant) and cABL genes did not increase during the storage period. RNA integrity number (RIN) indicated the degradation of RNA during FFPE processing, although no further significant degradation occurred during the course of the experiment. Conclusions: The results of this study indicated that although the RNA was impacted by the tissue preparation, fixation, and processing steps, for the brain FFPE slide specimens, target genes with amplicon size up to 124bp could be detected with minimum degradation for up to 12 months when slides were stored at room temperature.

scholarly journals Stabilization of circulating thyroglobulin mRNA transcripts in patients treated for differentiated thyroid carcinoma

2016 ◽  
Vol 54 (5) ◽  
pp. 558-566 ◽  
Author(s):  
Søren Feddersen ◽  
Lars Bastholt ◽  
Susanne M Pedersen
Keyword(s):  
Thyroid Carcinoma ◽  
Real Time ◽  
Real Time Pcr ◽  
Room Temperature ◽  
Differentiated Thyroid Carcinoma ◽  
Mrna Levels ◽  
Serum Thyroglobulin ◽  
Tempus Tubes ◽  
Antithyroglobulin Antibodies ◽  
Previously Treated

Background The clinical utility of serum thyroglobulin in the follow-up of patients with differentiated thyroid carcinoma may be compromised by the presence of endogenous antithyroglobulin antibodies. To prevent interference by antithyroglobulin antibodies several groups have developed real-time PCR-based assays for quantification of blood thyroglobulin mRNA levels. For accurate quantification of thyroglobulin mRNA in blood preanalytical factors must be recognized and controlled. In this study, we evaluate the effect of different blood RNA stabilizing systems – the Tempus Blood RNA system and the PAXgene Blood RNA system – and storage time on RNA yield and quality, and thyroglobulin mRNA stability. Methods Blood samples from 11 patients previously treated for differentiated thyroid carcinoma were collected in K2-EDTA, Tempus and PAXgene tubes and maintained at room temperature. RNA was isolated following storage for 0 and 72 h, and RNA yield, integrity and purity was determined. Thyroglobulin, GAPDH and ACTB mRNA levels were quantified by semi-quantitative real-time PCR. Results The RNA yield was significantly higher for blood collected in Tempus tubes compared with PAXgene tubes following storage for 72 h at room temperature ( P = 0.0011). High-quality RNA could be extracted from blood collected in PAXgene and Tempus tubes. Blood collected in K2-EDTA tubes, but not in PAXgene and Tempus tubes, showed significant changes in thyroglobulin mRNA levels following storage for 72 h at room temperature ( P = 0.0263). Conclusions Stabilization of blood in PAXgene and Tempus tubes enables storage at room temperature for up to 72 h, without compromising thyroglobulin mRNA levels.

Screening of NSCLC samples from Chinese lung cancer patients for activating rearrangements of the ALK, RET, and ROS1 genes.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e22066-e22066
Author(s):  
Li-Mou Zheng ◽  
David B. Whyte ◽  
Li Ruan ◽  
Roman Song ◽  
Luo Fei ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Real Time ◽  
Real Time Pcr ◽  
Kinase Inhibitors ◽  
Ffpe Tissue ◽  
Cancer Tissue ◽  
Fusion Genes ◽  
Tissue Samples ◽  
Lung Cancers ◽  
Pcr Assays

e22066 Background: The ALK, RET, and ROS1 genes are involved in gene rearrangements in a fraction of non-small cell lung cancers. The resulting oncogenic fusion genes define molecular sub-types of NSCLC with distinct sensitivities to treatment with various kinase inhibitors. We developed real-time reverse transcriptase PCR assays to detect rearrangements of ALK, RET, and ROS1 in FFPE lung cancer tissue. Methods: mRNA from NSCLC FFPE tissue samples was reverse transcribed to cDNA. Multiplex quantitative PCR was performed to detect 9 variants of EML4-ALK fusions, 9 variants of RET fusions and 14 variants of ROS1 fusions. A total of 409 samples were analyzed: 267 were classified as adenocarcinoma, 104 as squamous cell carcinoma and 38 had undetermined histology. EGFR and KRAS mutation status is unknown. The junctions of fusion-positive samples were sequenced by Sanger sequencing. Results: Among the 409 NSCLC specimens tested the frequency was 5.4% (22/409) for EML4-ALK fusions, 1.5% (6/409) for RET fusions, and 2.2% (9/409) for ROS1 fusions. EML4-ALK fusions were more prevalent in patients that were less than 60 years old (9.1% versus 2.0%, p= 0.004). The TNM stage was not correlated with the presence of any of the fusions. The table below lists the frequencies for specific rearrangements as determined by sequencing the real-time PCR products. Conclusions: Real-time PCR assays based on cDNA from FFPE tissue can identify patients with ALK, RET and ROS1 fusion genes. The ALK, RET and ROS1 assays will allow selection of patients most likely to respond to therapies that specifically target these cancer drivers. Further clinical testing of NSCLC patients in the Chinese population will be performed to support SFDA registration of these assays in China. [Table: see text]

Prognostic Value of JAK2V617F, Calr and MPL Mutational Status on Outcome and Thrombotic Risk in a Retrospective Cohort of 138 Patients with Essential Thrombocythemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5553-5553
Author(s):  
Giulia Benevolo ◽  
Ludovica Riera ◽  
Barbara Nicolino ◽  
Andrea Evangelista ◽  
Eloise Beggiato ◽  
...  
Keyword(s):  
Overall Survival ◽  
Real Time ◽  
Essential Thrombocythemia ◽  
Real Time Pcr ◽  
Cumulative Incidence ◽  
Retrospective Cohort ◽  
Prognostic Value ◽  
Thrombotic Risk ◽  
Activating Mutations ◽  
Mutational Status

Abstract Background: Life expectancy in Essential Thrombocythemia (ET) patients is superimposed to normal population. The main causes of death are thrombotic events and evolution into myelofibrotic phase or secondary myelodisplasia/acute leukemia. Approximately 50% to 65% of patients with ET carry activating mutations in the Janus kinase 2 gene (JAK2), and an additional 5% in the thrombopoietin receptor gene (MPL) whereas 10 to 20% of patients have mutated calreticulin gene (CALR). Non-mutated JAK2, CALR and MPL ET (triple negative-TN) are about 10%. Patients and methods : In this study we analysed the prognostic value of JAK2V617F, CALR and MPL mutational status on outcome and thrombotic risk in a retrospective cohort of 138 ET patients defined according to WHO criteria, diagnosed from 1974 to 2013 in a single Italian centre (Turin). JAK2V617F mutation was assessed by Quantitative Real–Time PCR on DNA from peripheral blood or bone marrow samples. JAK2 negative cases were then analyzed for CALR mutations by Gene Scan Analysis in combination with direct sequencing or MPL W515L/K by Allelic Discrimination Real-Time PCR assay. Overall survival (OS), cumulative incidence of myelofibrotic transformations (CI-MT) and thrombosis (CI-TB) were calculated from the date of diagnosis. The between-group comparison for OS was performed with the log-rank test, whereas for CI-MT and CI-TB we using the Gray’s test considering death as competing event and adjusting for presence of cardiovascular risk factors . Results: Among 138 ET patients, 103 (74.6%) carried the JAK2V617F mutation, 3 (2.2%) carried activating mutations of MPL exon 10, 16 (11.6%) carried mutations of CALR exon 9, and only 16 patients (11.6%) had none of these markers (TN). An high incidence of elevated haemoglobin levels and/or haematocrit (males >16.5 g/dl or 49% and females >16.0 g/dl or 48%) was significantly associated with JAK2 positivity [13 pts JAK2+ (14.77%) vs 0 pts JAK2- (Fisher test p=0.019)]. Similarly, the CI-TB, analysed with a competing-risk approach, was higher in patients with a JAK2 mutation [(5-year CI-TB: 23.7% JAK2, 0% CALR, 0% MPL, 12.5% TN; P<0.001)]. With a median follow-up of 48 months (IQR: 27-78; range 1-269), we observed 4 (3%) deaths, 9 (7%) myelofibrotic transformations and none leukemic evolution. The overall survival at 5 years was 98.4% (95%CI: 89.1-99.8) and the cumulative incidence of myelofibrotic transformations was 2.1% (95%IC: 1.0-8.6). The CI-MT was significantly higher in MPL-positive patients [5-year CI-MT: 0% JAK2, 0% CALR, 33.3% MPL, 8.3% TN; (P=0.006)]. Not significant difference on OS was found [5-year OS: 100% JAK2, 100% CALR, 100% MPL, 85.7% TN; (P=0.66)]. We did not include leukemia transformation, because of lack of event. Conclusion: In our retrospective cohort of ET patients, we found a significant correlation between JAK2 positivity and high risk of thrombosis. We also found a significant incidence of elevated haemoglobin levels and/or haematocrit in JAK2-positive patients. These data support the correlation between JAK2 positivity and cumulative risk of transformation to Polycythemia Vera. The correlation between MPL mutational status and evolution to myelofibrosis found in our study needs to be confirmed in a larger series of patients. Disclosures No relevant conflicts of interest to declare.

Rapid real-time PCR for detection of Mycobacterium tuberculosis complex DNA in formalin-fixed paraffin embedded tissues: 16% of histological ‘sarcoid’ may contain such DNA

2014 ◽  
Vol 67 (12) ◽  
pp. 1084-1087 ◽  
Author(s):  
Güzin Surat ◽  
William A Wallace ◽  
Ian F Laurenson ◽  
Amie-Louise Seagar
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Extraction Methods ◽  
Ffpe Tissue ◽  
Negative Results ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Dna Extraction Methods ◽  
Formalin Fixed

AimsTo investigate the diagnostic accuracy of IS6110 real-time PCR for detection of Mycobacterium tuberculosis complex (MTBC) in DNA extracted from formalin-fixed paraffin embedded (FFPE) tissues using two different methods. In the absence of material submitted for tuberculosis (TB) culture, MTBC detection in FFPE tissue can be an important aid to diagnosis.MethodsWe collected 144 FFPE tissue blocks (lung and lymph node) for IS6110 real-time PCR. Two DNA extraction methods (QIAamp FFPE tissue kit and NucliSENS easyMAG) were assessed within a general laboratory setting. PCR results were compared with histology and culture.ResultsIn the histological MTBC and culture MTBC (TB-positive) groups, 72.4% were IS6110-positive and 27.6% negative. IS6110-negative results were obtained from 98%, 61.5% and 84% of the histologically MTBC-negative (TB-negative) group, histologically TB/no culture group and sarcoidosis group, respectively. Review of 19 IS6110-positive patients in the latter three groups showed that 15 had clinical TB. Thirteen of 15 (86.7%) IS6110-positive patients in the histological TB/no culture group and 2 of 4 (50%) IS6110-positive patients in the sarcoidosis group were clinically diagnosed with TB which highlights the difficulty of a pathological diagnosis.ConclusionsIS6110 real-time PCR using easyMAG extracted DNA is a moderately sensitive, specific and rapid method for MTBC detection in FFPE material, but must be interpreted in the overall clinical context. PCR results can be available in around 5 h from FFPE specimen receipt, with minimal hands-on time.

DNMT3A is a Powerful Follow-up Marker in NPM1 mutated AML

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 122-122 ◽  
Author(s):  
Susanne Schnittger ◽  
Claudia Haferlach ◽  
Tamara Alpermann ◽  
Niroshan Nadarajah ◽  
Manja Meggendorfer ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Melting Curve Analysis ◽  
Employment Equity ◽  
Low Level ◽  
Mutational Status ◽  
Equity Ownership ◽  
The Stability

Abstract Background: NPM1 mutated (mut) FLT3-ITD negative acute myeloid leukemia (AML) is a distinct prognostically favorable subtype of AML. Robust data is available demonstrating that monitoring therapy response using NPM1mut-specific real time PCR is an important tool to early detect relapses and provides important information to guide therapy. Since next generation sequencing techniques have become available further gene mutations were detected that accompany NPM1mut in AML. Of these DNMT3Amut were the most frequent and stable ones (Krönke et al., Blood, 2013). Aim: 1) Analyse the stability of DNMT3Amut in paired diagnostic and relapsed samples. 2) Evaluate whether monitoring of DNMT3Amut provides additional information to monitoring of NPM1mut. Patients and Methods: Samples were selected from a cohort of 359 NPM1mut de novo AML cases with an available DNMT3Amut status.First, toevaluate the stability of DNMT3Amut paired diagnostic and relapse samples of 103 patients were analyzed (44 males, 59 females; median age 60 years, range: 26-82 years). Median time to relapse was 11 months (range: 3-68 months). NPM1mut status was assessed at diagnosis with a LightCycler melting curve analysis assay. Non type A mutations were further characterized by Sanger sequencing. Second, all diagnostic and follow-up samples (n=1,813) were quantified by real time PCR specific for the individual NPM1mut. Analysis for DNMT3Amut was performed using either the 454 technology (454 Life Sciences, Branford, CT) or the MiSeq instrument (Illumina, San Diego, CA). Deep DNMT3A sequencing of remission samples was performed using the 454 technology. Results: Out of 103 paired samples 61 (59.2%) carried a DNMT3Amut at diagnosis. 57/61 (93.4%) patients stably retained the mutation at relapse, in 4 (6.6%) the DNMT3Amut was lost. On the other hand 2 of 42 (4.8%) cases with DNMT3A wildtype at diagnosis gained the mutation at relapse. Thus, DNMT3Amut status was shown to be relatively stable (97/103; 94.1%) and thus qualifies as a promising target for follow-up controls. For comparison of DNMT3Amut and NPM1mut status during follow-up 54 patients that were NPM1/DNMT3A double mutated at diagnosis were selected according to the availability of at least one sample in first remission with an NPM1mut level <0.01%. These samples were reanalyzed by deep sequencing for the respective DNMT3A amplicons that had identified mutations at diagnosis. Two of these 54 cases (3.7%) showed morphologic relapse but NPM1mut was negative at relapse (sensitivity of 10-7). In one of these two cases at diagnosis NPM1mut and TET2mut were observed while at relapse IDH1mut and RUNX1mut were present. However, at both time points the DNMT3Amut was identified. The second case lost NPM1mut and CEBPAmut and retained the DNMT3Amut and TET2mut. Thus in these 2 cases the DNMT3Amut can be regarded as the common ancestor. 1 case retained the NPM1mut at relapse but lost the DNMT3Amut. Of note, in 32/54 (59.3%) cases the DNMT3Amut persisted in the remission samples (NPM1mut low level <0.01% or negative) with high DNMT3Amut loads (median: 20%, range: 2-59%) that was only slightly below the load at diagnosis (median: 45%, range: 38-58%). To analyze the clinical importance of these persisting DNMT3Amut survival analysis was performed. Median overall survival for patients with persisting DNMT3Amut (n=32) was 69 vs 96 months in those who lost also the DNMT3Amut in remission (n=22, p=0.053). Median event free survival was 38 vs. 96 months (p=0.031). Thus the DNMT3A mutational status in remission of NPM1mut AML is a further important parameter for prognostication. The mechanisms underlying this observation are obscure. As NPM1mut disappeared in remission and DNMT3A was retained and with the exception of 2 cases all others (n=18) relapsed with an NPM1mut (even the same type as at diagnosis) two mechanisms may be discussed: 1) Persisting DNMT3Amut cells predispose by a yet unknown mechanisms to the development of a secondary NPM1mut or 2) a residual DNMT3Amut/NPM1mut very low level survivor is able to overgrow the DNMT3Amut sole mutated clone in remission and cause relapse. Conclusions: 1) DNMT3Amut persist in remission of NPM1mut AML in the majority of cases (59.2 %). 2) DNMT3Amut analysis in remission of NPM1mut AML is an important parameter for prognostication. 3) Clones with DNMT3Amut as the sole mutation may have a normal phenotype and thus DNMT3Amut may even be regarded as premalignant mutation. Disclosures Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

A Real-time PCR Assay for the Detection of EGFR Mutations in FFPE Tissue and Plasma Samples of NSCLC Patients.

2016 ◽  
Vol 34 (15_suppl) ◽  
pp. e23251-e23251 ◽  
Author(s):  
Patrick O'Donnell ◽  
Johnny Shyu ◽  
Jane Ferguson ◽  
Kelli Demartin ◽  
Theresa May ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Ffpe Tissue ◽  
Egfr Mutations ◽  
Plasma Samples ◽  
Pcr Assay ◽  
Nsclc Patients

KRAS aKtive, an Italian network for assessment of KRAS mutations in colorectal cancer patients: Results on 7,432 cases.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e14042-e14042
Author(s):  
Antonio Marchetti ◽  
Carmine Pinto ◽  
Gian Luigi Taddei ◽  
Claudio Clemente ◽  
Giancarlo Troncone ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Real Time ◽  
Cancer Patients ◽  
Real Time Pcr ◽  
Accurate Estimation ◽  
Kras Mutations ◽  
Detection Techniques ◽  
Mutational Status ◽  
Colorectal Cancer Patients ◽  
Large Survey

e14042 Background: The KRAS aKtive program was started on March 2009, promoted by the Italian Association of Medical Oncology (AIOM) and the Italian Society of Surgical Pathology and Cytopathologyy (SIAPEC) to support the activity of oncologists and pathologists involved in the management of metastatic colorectal cancer patients who need the assessment of the mutational status of the KRAS gene. Methods: The program was specifically devised to facilitate the exchange of biologic material, clinicopathological data and diagnostic reports within a network of oncologist, pathologists and pathology/molecular biology reference laboratories throughout Italy, connected through the site www.kras-aKtive.it. KRAS mutation analysis was performed by Sanger sequencing (SS), real time PCR or other techniques, including pyrosequencing and hybridization strip assays. Data were collected in a common database. Results: The KRAS aKtive program has involved 478 oncologists, 144 pathologists, and 24 reference laboratories. A total of 7,432 KRAS mutations analyses were performed. The tests were informative in 7,265 cases (98%). The vast majority of tests (5,626 cases, 77.4%) were conducted by SS. In 529 (7.3%) cases a real-time PCR assay was used, other detection techniques were used in 1,110 (15.3%) cases. KRAS mutations at codons 12-13 were detected in 2,874 cases (39,6%). The frequency of mutations detected by real-time PCR or other techniques (45%, and 43%, respectively) was significantly higher (p=0.002, and p=0.008%, respectively) than that observed by SS (38%). The percentage of cases evaluated by non-SS-based methods has increased during the first three years of the program. Conclusions: The results of this large survey allow an accurate estimation of the actual prevalence of KRAS mutations and their types in caucasian colorectal cancer patients. Our data indicate that the frequency of mutations detected by non-SS-based methods is higher than that obtained by SS.

scholarly journals ANALISIS PENGARUH PAPARAN FISIK PADA SAMPEL GIGI TERHADAP HASIL KUANTIFIKASI DNA FORENSIK MENGGUNAKAN METODE KIT PURIFIKASI DNA KOMERSIAL

2021 ◽  
Vol 5 (2) ◽  
pp. 59-65
Author(s):  
Septi Arini ◽  
Arief Budi Witarto ◽  
Setia Betaria Aritonang
Keyword(s):  
Real Time ◽  
River Water ◽  
Real Time Pcr ◽  
Room Temperature ◽  
Sea Water ◽  
Water Immersion ◽  
Dna Quantification ◽  
Forensic Dna ◽  
Physical Exposure ◽  
Free Air

Physical exposure to biological samples has an enormous influence on the results of forensic DNA analysis. The lack of molecular research on the effect of physical exposure on dental samples is the reason for the need for further research. This study aims to determine the effect of physical exposure on dental samples on the results of forensic DNA quantification. The parameter used is the concentration value of isolated DNA obtained from real time PCR analysis. The use of real time PCR allows the detection and quantification of specific sequences of DNA samples at the same time to be analyzed. The dental samples used were obtained from different individuals. Teeth are used as identification media because teeth are the hardest part of the body and are chemically the most stable and most resistant to degradation and decomposition. The method in this study is to give three types of treatment to the tested samples in the form of sea water immersion, river water immersion and exposure to free air at room temperature with each treatment consisting of three test samples. All samples were extracted using a Commercial DNA purification KIT with a reagent in the form of a Qiagen KIT (QIAamp® DNA Investigator) then a quantification process was carried out to see the value of the DNA concentration of each sample using real time PCR. The results of DNA quantification of dental samples from each treatment showed that the highest sample concentration value was based on the average of each treatment, namely samples with treatments exposed to free air at room temperature with a concentration value of 1.34 ng/µl, followed by samples soaked using river water with a concentration value of 0.15 ng/µl, while the sample with the lowest concentration is shown by a sample treated with seawater immersion with a concentration value of 0.10 ng/µl. Physical exposure in the form of exposure to free air, exposure to river water and exposure to sea water on dental samples, gave a not too significant effect on the results of DNA quantification produced.

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