Increased levels of HPV16 E6*I transcripts in high-grade cervical cytology and histology (CIN II+) detected by rapid real-time RT-PCR amplification

The purpose of this study was to determine whether Toll-like receptor 5 (TLR5) expression was associated with disease progression in cervical neoplasia. TLR5 expression was evaluated by immunohistochemistry (IHC) in 55 formalin-fixed paraffin-embedded cervical tissues; 10 normal cervical specimens, 9 low-grade cervical intraepithelial neoplasias (CINs), 12 high-grade CINs, and 24 invasive squamous cell carcinomas (ISCCs). TLR5 expression was also evaluated at the RNA level, in fresh, frozen cervical carcinoma tissues by real-time quantitative RT-PCR. TLR5 expression, which was mainly observed as cytoplasmic staining, gradually increased in accordance with the histopathologic grade in the following order: low-grade CIN less than high-grade CIN less than ISCC (P< 0.001). Immunohistochemical staining showed that TLR5 expression was undetectable (80%) or weak (20%) in normal cervical squamous epithelial tissues. However, moderate expression was detected in 33.3% of low-grade CIN (3/9), 41.7% of high-grade CIN (5/12), and 45.8% of ISCC (11/24). Strong expression was detected in as much as 33.3% of high-grade CIN (4/12) and 50% of ISCC (12/24). Contrary to IHC results, real-time quantitative RT-PCR revealed that TLR5 expression in tumors was not statistically different compared to normal cervical tissues (P= 0.1452). The IHC result suggests that TLR5 may play a significant role in tumor progression of cervical neoplasia and may represent a useful marker for malignant transformation of cervical squamous cells.

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Real-time PCR provides evidence for thyrotropin receptor mRNA expression in orbital as well as in extraorbital tissues

Acta Endocrinologica ◽

10.1530/eje.0.1470733 ◽

2002 ◽

pp. 733-739 ◽

Cited By ~ 32

Author(s):

P Agretti ◽

L Chiovato ◽

G De Marco ◽

C Marcocci ◽

B Mazzi ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pcr Amplification ◽

Thyroid Diseases ◽

Variant Form ◽

Rt Pcr ◽

Specific Primers ◽

Beta Actin ◽

Tshr Gene ◽

Tissue Specimens

OBJECTIVE: The TSH receptor (TSHr) expressed on thyroid follicular cells is the autoantigen involved in the pathogenesis of Graves' hyperthyroidism. Whether this receptor is expressed in extrathyroidal tissues, and whether it participates directly in the pathogenesis of thyroid-associated ophthalmopathy (TAO) is unclear. DESIGN: The aim of the present study was to measure TSHr mRNA in retro-orbital tissues, retro-orbital adipose tissue, extraocular muscle, and skin from patients with TAO and in several tissues from patients not affected by thyroid diseases using RT-PCR and real-time PCR. METHODS: Total RNA was isolated from tissue specimens, reverse transcribed, and amplified using specific primers for the extracellular portion and a part of a 1.3 kbp variant form of the TSHr gene. Determination of TSHr mRNA levels using real-time PCR was also performed by the TaqMan system; to normalize for differences in the amount of total RNA added to the reaction, amplification of beta-actin gene was performed as an endogenous control. RESULTS: A single-round RT-PCR amplification using specific primers for the extracellular portion of the TSHr gene demonstrated an amplification product of 1.2 kbp in the thyroid, but not in all other tissues. A second-round RT-PCR amplification using the same primers and starting from the previous amplification product demonstrated a band of the size expected for the TSHr gene in all tissue specimens analyzed irrespective of their origin. Similar results were obtained using primers specific for a part of the variant form of 1.3 kbp of the TSHr gene. The amount of TSHr mRNA measured by real-time PCR with the TaqMan probe and expressed as TSHr/beta-actin ratio was similar in the tissues from TAO patients with respect to the tissues from subjects not affected by thyroid diseases. CONCLUSIONS: We measured TSHr mRNA in tissues from patients with TAO using the very sensitive and quantitative method of real-time PCR. The level of transcription was similar to that measured in extraorbital tissues from patients who were not affected by thyroid diseases. These data suggest an illegitimate TSHr mRNA transcription in all the tissues examined apart from thyroid.

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Combination of HPV E6/E7 and HTERT mRNA real-time RT-PCR assay for diagnosing high grade cervical lesions and malignancy

Pathology ◽

10.1016/j.pathol.2015.12.405 ◽

2016 ◽

Vol 48 ◽

pp. S149

Author(s):

Sunyoung Park ◽

Hye-young Wang ◽

Sunghyun Kim ◽

Geehyuk Kim ◽

Dongsup Lee ◽

...

Keyword(s):

Real Time ◽

High Grade ◽

Htert Mrna ◽

Rt Pcr ◽

Cervical Lesions ◽

Pcr Assay ◽

Hpv E6

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Real-time quantitative RT-PCR for detection of the HPV16-E6 and -E7 genes

Otolaryngology ◽

10.1016/s0194-5998(03)01111-2 ◽

2003 ◽

Vol 129 (2) ◽

pp. P190

Author(s):

M Underbrink

Keyword(s):

Real Time ◽

Rt Pcr ◽

Hpv16 E6 ◽

E6 And E7

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LB1.66 Detection of zika virus and cytomegalovirus in cervical cytology samples of pregnant women from guayaquil, ecuador, using two real-time polymerase chain reaction (RT-PCR) molecular assays

10.1136/sextrans-2017-053264.171 ◽

2017 ◽

Author(s):

Héctor Zambrano ◽

Jesse Waggoner ◽

Karina León ◽

Benjamin Pinsky ◽

Marissa Schettino ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Pregnant Women ◽

Real Time ◽

Zika Virus ◽

Cervical Cytology ◽

Rt Pcr ◽

Chain Reaction ◽

Molecular Assays ◽

Polymerase Chain

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Robust CT-prediction algorithm for RT-PCR

Filomat ◽

10.2298/fil1604103g ◽

2016 ◽

Vol 30 (4) ◽

pp. 1103-1110 ◽

Cited By ~ 1

Author(s):

Melih Gunay ◽

Rajarajeswari Balasubramaniyan

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pcr Amplification ◽

Prediction Algorithm ◽

Rt Pcr ◽

Ct Value ◽

Alternative Approach ◽

Definition Of ◽

Multiple Pathogens

Introduction of fluorescence-based Real-Time PCR (RT-PCR) also called qPCR is increasingly used to detect multiple pathogens simultaneously and rapidly by gene expression analysis of PCR amplification data. Real-time PCR data are analyzed after setting an arbitrary threshold that must intersect the signal curve in its exponential phase. The point at which the curve crosses the threshold is called CT (Cycle Threshold). This simple and arbitrary value however is not an elegant definition of CT value sometimes leads to conclusions that are either false positive or negative. Therefore, the purpose of this paper is to present a stable and consistent alternative approach for the definition and determination of CT value that leads to near zero false positives and negatives.

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Selection of Housekeeping Genes for Transgene Expression Analysis in Eucommia ulmoides Oliver Using Real-Time RT-PCR

Journal of Botany ◽

10.1155/2010/230961 ◽

2010 ◽

Vol 2010 ◽

pp. 1-7 ◽

Cited By ~ 10

Author(s):

Ren Chen ◽

Mayumi Gyokusen ◽

Yoshihisa Nakazawa ◽

Koichiro Gyokusen

Keyword(s):

Gene Expression ◽

Real Time ◽

Expression Analysis ◽

Transgene Expression ◽

Pcr Amplification ◽

Housekeeping Genes ◽

Housekeeping Gene ◽

Amplification Efficiency ◽

Eucommia Ulmoides ◽

Rt Pcr

In order to select appropriate housekeeping genes for accurate calibration of experimental variations in real-time (RT-) PCR results in transgene expression analysis, particularly with respect to the influence of transgene on stability of endogenous housekeeping gene expression in transgenic plants, we outline a reliable strategy to identify the optimal housekeeping genes from a set of candidates by combining statistical analyses of their (RT-) PCR amplification efficiency, gene expression stability, and transgene influences. We used the strategy to select two genes, ACTα and EF1α, from 10 candidate housekeeping genes, as the optimal housekeeping genes to evaluate transgenic Eucommia ulmoides Oliver root lines overexpressing IPPI or FPPS1 genes, which are involved in isoprenoid biosynthesis.

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Seroprevalence and Real-time PCR Detection of Brucellosis in Abattoirs Animals as a Potential Route of Infection in Egypt

European Journal of Medical and Health Sciences ◽

10.24018/ejmed.2019.1.5.105 ◽

2019 ◽

Vol 1 (5) ◽

Author(s):

Amira Mohamed Zakaria ◽

Salwa F. Ahmed ◽

Mohamed S. Motawae

Keyword(s):

Real Time ◽

Rose Bengal ◽

Real Time Pcr ◽

Serological Test ◽

Pcr Amplification ◽

Cost Effective ◽

Rt Pcr ◽

Serum Samples ◽

Molecular Approaches ◽

Rose Bengal Test

Brucellosis is among the most common and economically serious zoonosis worldwide. Brucellosis in Egypt is an endemic problem among animals and humans. This work intended to evaluate the conventional serological and molecular approaches as a tool for studying the prevalence of brucellosis within abattoir’s animals in two large Egyptian provinces. Two hundred and thirty (n=230) blood and serum samples were collected from (2-3) years male calves in two Egyptian abattoirs. Rose Bengal test (RBT) and modified in-house ELISA were applied to determine the seroprevalence of Brucellosis in abattoirs animals while quantitative Taqman real-time PCRs (RT-PCR) were implemented for the characterization of Brucella species. The overall prevalence of brucellosis in the two proveinces was (53.9 %) , (75.2 %) and (79.1 %) as determined by ELISA ,RBT and RT- PCR assays respectively. Brucella DNA was successfully amplified from serum samples as well as blood. A total of n= 182 samples (79.1 %) were identified by real time PCR amplification for IS711 gene as Brucella genus, n= 118 (64.8 %) were reported as B. aborts while n= 85 (46.7 %) were reported as B. melitensis. N= 44 (24.17 %) from the collected samples comprised the two species of bacteria. This study endorses the application of rose Bengal test as a sensitive and cost effective serological test for brucellosis and real-time PCR as a distinguishing tool to detect the causative agents. Our findings indicate a significantly high prevalence of Brucella antibodies and DNA in blood and serum samples which poses a crucial threat to public health in Egypt.

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