scholarly journals Molecular Origins of Bimodal mRNA Copy Number Distribution

2015 ◽  
Vol 108 (2) ◽  
pp. 534a
Author(s):  
Keisuke Fujita ◽  
Mitsuhiro Iwaki ◽  
Toshio Yanagida
Keyword(s):  
Copy Number ◽  
Mrna Copy Number ◽  
Number Distribution

Immune gene expression in primary melanomas to predict lower risk of recurrence and death.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 3014-3014
Author(s):  
Shanthi Sivendran ◽  
Rui R. Chang ◽  
Sara Harcharik ◽  
Lawrence Hall ◽  
Sebastian Bernardo ◽  
...  
Keyword(s):  
Copy Number ◽  
Roc Curves ◽  
Gene Panel ◽  
Prolonged Survival ◽  
Immune Gene ◽  
Training Cohort ◽  
Patient Cohort ◽  
Mrna Copy Number ◽  
Immune Related Genes ◽  
The Impact

3014 Background: Improved biomarkers are needed to define recurrence risk in patients with completely resected skin melanomas. Standard prognostic indicators used in staging, including depth, ulceration, and mitotic rate, while useful, often fail to accurately predict recurrence for individual patients. The immune system may prevent recurrence in this population, but no evidence-based immune biomarkers are in clinical use. Biomarker development has been hindered by clinical standards necessitating that the entire specimen be formalin fixed and paraffin embedded (FFPE) for morphology evaluation, a process damaging to RNA. Methods: To define a biomarker for melanoma recurrence, mRNA copy number of immune-related genes from FFPE melanoma was measured using NanoString, a hybridization assay suited for analysis of partially degraded RNA. Genes predictive of non-recurrence were defined using receiver operating characteristic (ROC) curves in a training cohort and then validated in an independent patient cohort. Results: A panel of 21 genes predictive of non-recurrence were defined using ROC curves in a training cohort (N=44). This result was validated in an independent patient cohort (N=37, AUC=0.794). Protein levels of the most differentially expressed gene, CD2, also associated with non-recurrence (p<0.001). The immune gene panel and CD2 staining associated with prolonged survival (p<0.001 and p=0.019, respectively). Conclusions: mRNA copy number of immune-related genes in primary FFPE melanomas predicts non-recurrence and prolonged survival. This data highlights the impact of immunosurveillance in primary human melanoma and the identified gene panel may be a useful tool for patient stratification for adjuvant immunotherapy studies.

scholarly journals Two variants of quantitative reverse transcriptase PCR used to show differential expression of α-, β- and γ-fibrinogen genes in rat liver lobes

1997 ◽  
Vol 321 (3) ◽  
pp. 769-776 ◽  
Author(s):  
Junlong ZHANG ◽  
Mina DESAI ◽  
Susan E. OZANNE ◽  
Cora DOHERTY ◽  
C. Nicholas HALES ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Rat Liver ◽  
Coefficient Of Variation ◽  
Copy Number ◽  
Internal Standard ◽  
Rt Pcr ◽  
Total Rna ◽  
Reverse Transcriptase Pcr ◽  
Mrna Copy Number ◽  
Sensitive Method

Quantitative reverse transcriptase PCR (RT-PCR) is a sensitive method for the measurement of mRNA copy number. However, the methodology has gained a reputation for poor reproducibility, leading to concern over the validity of much of the data generated using this technique. We have developed two variants of quantitative competitive RT-PCR using a synthesized RNA as an internal standard to measure precisely the relative levels of α-, β- and γ-fibrinogen mRNAs in the four lobes of the rat liver. In the first of these variants we altered only the amount of total RNA in the RT-PCR reaction, keeping the amount of internal standard RNA and the number of PCR cycles constant. In the second variant only the number of PCR cycles was altered, and the amounts of total RNA and standard RNA were kept constant. Both variants of RT-PCR allowed calculation of the number of mRNA copies, which did not differ significantly between the two techniques. Of the two variants, the second gave better reproducibility, and the intra-assay coefficient of variation for this technique was 14% (n = 20). Using these two variants we have shown that there are different numbers of fibrinogen mRNAs in the four liver lobes for each of the three genes (α-fibrinogen F = 14.64, P = 0.0003; β-fibrinogen F = 3.74, P = 0.04; γ-fibrinogen F = 3.75, P = 0.04). In conclusion, by using two variants of quantitative competitive RT-PCR we have shown that this technique can be used to give reproducible results, and the low intra-assay coefficient of variation suggests that quantitative RT-PCR should be the technique of choice for accurate measurement of mRNA copy number.

scholarly journals Survivin mRNA Copy Number in Bladder Washings Predicts Tumor Recurrence in Patients with Superficial Urothelial Cell Carcinomas

2004 ◽  
Vol 50 (8) ◽  
pp. 1425-1428 ◽  
Author(s):  
Iman J Schultz ◽  
Lambertus A Kiemeney ◽  
Herbert F M Karthaus ◽  
J Alfred Witjes ◽  
Johannes L Willems ◽  
...  
Keyword(s):  
Copy Number ◽  
Tumor Recurrence ◽  
Urothelial Cell ◽  
Survivin Mrna ◽  
Mrna Copy Number

scholarly journals Differential priming of RNA templates during cDNA synthesis markedly affects both accuracy and reproducibility of quantitative competitive reverse-transcriptase PCR

1999 ◽  
Vol 337 (2) ◽  
pp. 231-241 ◽  
Author(s):  
Junlong ZHANG ◽  
Christopher D. BYRNE
Keyword(s):  
Reverse Transcriptase ◽  
Copy Number ◽  
Accurate Determination ◽  
Fold Increase ◽  
Pcr Primers ◽  
Cdna Synthesis ◽  
Reverse Transcriptase Pcr ◽  
Target Mrna ◽  
Mrna Copy Number ◽  
Synthesis Reactions

Quantitative competitive reverse-transcriptase PCR is the most sensitive method for studying gene expression. To investigate whether the accuracy of the calculated target mRNA copy number is affected by the cDNA priming process, we utilized primers of different lengths, concentrations and primer sequences to prime cDNA synthesis reactions. Our results show a ≈ 19-fold increase in the calculated mRNA copy number from cDNA synthesis reactions primed with random hexamers (P< 0.001, n = 4), and a ≈ 4-fold increase in copy number with a specific hexamer (P< 0.001, n = 4) compared with that obtained with a 22-mer-sequence-specific primer. The increase in calculated mRNA copy number obtained by priming cDNA synthesis with the shorter specific and non-specific primers could be explained largely by the synthesis of truncated standard cDNA molecules lacking a requisite binding site for amplification with PCR primers. Since these truncated standard cDNA molecules could not be amplified and standard RNA is used to quantify target mRNA copy number, this phenomenon resulted in overestimation of target mRNA copy number. In conclusion, accurate determination of target mRNA copy number is most likely if a long specific antisense primer is used to prime cDNA synthesis.

scholarly journals Resveratrol affects Zika virus replication in vitro

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Azirah Mohd ◽  
Nurhafiza Zainal ◽  
Kim-Kee Tan ◽  
Sazaly AbuBakar
Keyword(s):  
Cell Cultures ◽  
Zika Virus ◽  
Copy Number ◽  
Virus Titer ◽  
Health Concern ◽  
Dependent Manner ◽  
Zikv Infection ◽  
Mrna Copy Number ◽  
Short Period

Abstract Zika virus (ZIKV) infection is a serious public health concern. ZIKV infection has been associated with increased occurrences of microcephaly among newborns and incidences of Guillain-Barré syndrome among adults. No specific therapeutics or vaccines are currently available to treat and protect against ZIKV infection. Here, a plant-secreted phytoalexin, resveratrol (RES), was investigated for its ability to inhibit ZIKV replication in vitro. Several RES treatment regimens were used. The ZIKV titers of mock- and RES-treated infected cell cultures were determined using the focus-forming assay and the Zika mRNA copy number as determined using qRT-PCR. Our results suggested that RES treatment reduced ZIKV titers in a dose-dependent manner. A reduction of >90% of virus titer and ZIKV mRNA copy number was achieved when infected cells were treated with 80 µM of RES post-infection. Pre-incubation of the virus with 80 µM RES showed >30% reduction in ZIKV titers and ZIKV mRNA copy number, implying potential direct virucidal effects of RES against the virus. The RES treatment reduced >70% virus titer in the anti-adsorption assay, suggesting the possibility that RES also interferes with ZIKV binding. However, there was no significant decrease in ZIKV titer when a short-period of RES treatment was applied to cells before ZIKV infection (pre-infection) and after the virus bound to the cells (virus internalization inhibition), implying that RES acts through its continuous presence in the cell cultures after virus infection. Overall, our results suggested that RES exhibited direct virucidal activity against ZIKV and possessed anti-ZIKV replication properties, highlighting the need for further exploration of RES as a potential antiviral molecule against ZIKV infection.

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