Qualitative and Quantitative Assessment of Modified In Situ Reverse Transcription-Polymerase Chain Reaction (In Situ RT-PCR) Method.

In situ reverse transcription-polymerase chain reaction (RT-PCR) was used in young leaves (from trees and in vitro shoots) and flower buds of almond (Prunus dulcis), a stone fruit, for cellular location of Prune dwarf virus (PDV, a member of the genus Ilarvirus). Sections obtained from samples fixed in formaldehyde and embedded in paraffin were refixed in formaldehyde to increase tissue preservation in the RT-PCR steps. The coat protein gene of PDV was used as the target to produce a cDNA copy that was amplified by PCR and visualized using a direct detection method with digoxigenin-labeled nucleotides. Protein digestion, PCR, and detection strategies were optimized for increased tissue preservation and signal intensity. PDV was found in infected samples within the vascular tissue of young leaves and flower buds as well as in the mesophyll in developing floral organs and in the generative and vegetative cells of pollen grains. PDV signals were observed in a ring surrounding the nucleus and spread in the cytoplasm. The results obtained are discussed in terms of the technique optimization and PDV distribution in tissues and transmission through pollen. The optimized protocol of in situ RT-PCR is a powerful technique to reveal low-abundant RNA species. Therefore, it is appropriate to study cell and subcellular distribution of RNA viruses in woody species.

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Demonstration of Cytoplasmic Tyrosinase mRNA in Tissue-Cultured Cells by Reverse Transcription (RT) In Situ Polymerase Chain Reaction (PCR) and RT PCR In Situ Hybridization

Diagnostic Molecular Pathology ◽

10.1097/00019606-199702000-00005 ◽

1997 ◽

Vol 6 (1) ◽

pp. 26-33 ◽

Cited By ~ 12

Author(s):

Pei-Xiang Li ◽

Lorna Cheng ◽

Duan-Ren Wen ◽

Paul B. Wissmann ◽

Jeanne Cheng ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

In Situ Hybridization ◽

Reverse Transcription ◽

Cultured Cells ◽

Rt Pcr ◽

Chain Reaction ◽

Polymerase Chain ◽

Pcr In Situ

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Comparison of Reverse Transcription-Polymerase Chain Reaction, Immunohistochemistry, and Fluorescence In Situ Hybridization Methodologies for Detection of Echinoderm Microtubule-Associated Proteinlike 4–Anaplastic Lymphoma Kinase Fusion–Positive Non–Small Cell Lung Carcinoma: Implications for Optimal Clinical Testing

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2011-0321-oa ◽

2012 ◽

Vol 136 (7) ◽

pp. 796-803 ◽

Cited By ~ 79

Author(s):

Michelle L. Wallander ◽

Katherine B. Geiersbach ◽

Sheryl R. Tripp ◽

Lester J. Layfield

Keyword(s):

Polymerase Chain Reaction ◽

Small Cell ◽

Wild Type ◽

Rt Pcr ◽

Small Cell Lung ◽

Chain Reaction ◽

Anaplastic Lymphoma ◽

Pcr Method ◽

Polymerase Chain

Context.—Echinoderm microtubule–associated proteinlike 4–anaplastic lymphoma kinase (EML4-ALK) gene fusions are detected in 3% to 13% of non–small cell lung carcinomas. Accurate testing for detection of EML4-ALK fusions is essential for appropriate therapy selection. Objective.—To compare reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH) methodologies for detection of EML4-ALK fusions. Design.—Forty-six pulmonary adenocarcinomas were selected with enrichment for wild-type epidermal growth factor receptor (EGFR) status (wild type, n = 42; mutant, n = 4). Specimens were tested by IHC (Dako; clone ALK1), FISH (Abbott Molecular; LSI ALK break apart), and RT-PCR (variants 1 and 3a/b). Results.—EML4-ALK variant 3a/b was detectable by RT-PCR, FISH, and IHC in 4% (2 of 46) of specimens. Complete agreement among FISH and IHC reviewers was obtained for variant 3a/b. No concordance existed among methodologies for the detection of EML4-ALK variant 1. The RT-PCR method detected variant 1 in 20% (9 of 46) of specimens. Agreement among FISH viewers was poor for variant 1 because only 11% (1/9) of specimens were scored as positive by all 3 viewers. The sensitivity of IHC for detection of variant 1 was also poor because only 1 of 9 samples (11%) was scored as positive. Overall, the frequency of EML4-ALK variants 1 and 3a/b was 24% (11 of 46) in adenocarcinomas enriched for wild-type EGFR status. One EML4-ALK variant 1 fusion was found to coexist with an EGFR exon 21 mutation. Conclusions.—The FISH interpretation demonstrated great variability among observers. The RT-PCR method was the most sensitive and least-subjective methodology for detection of EML4-ALK fusions.

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METHOD FOR FMD VIRUS 146S COMPONENT CONCENTRATION DETERMINATION WITH REAL-TIME REVERSE TRANSCRIPTION – POLYMERASE CHAIN REACTION IN VACCINE RAW MATERIALS

Veterinary Science Today ◽

10.29326/2304-196x-2018-2-25-31-35 ◽

2018 ◽

pp. 31-35

Author(s):

M. I. Doronin ◽

A. M. Timina ◽

D. A. Lozovoy ◽

V. A. Starikov ◽

D. V. Mikhalishin ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Regression Model ◽

Real Time ◽

Reverse Transcription ◽

Raw Materials ◽

Rt Pcr ◽

Chain Reaction ◽

Pcr Method ◽

Polymerase Chain

Method for determination of foot-and-mouth disease (FMD) virus 146S component concentration with real-time reverse transcription – polymerase chain reaction (rtRT-PCR) in vaccine raw materials is developed. Negative regression model of С146S = (30.269 – Ct)/4.155 type allowing determination of FMDV 146S particle concentrations based on the amplification threshold cycle values (Ct) is proposed. It has been experimentally proven that the quantity of the 146S component determined by the real-time RT-PCR method using developed regression model and contained in the inoculation dose of FMD vaccine confers protection to the vaccinated animals against generalized FMD of A, O, Asia-1 types. rtRT-PCR method is highly sensitive and allows rapid and highly reliable estimation of the 146S antigen concentration in FMD vaccine. The method for 146S particle quantity determination by real-time RT-PCR using the regression model is reliable and demonstrates high correlation (95.5–99.0%) with the complement fixation test results.

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Sensitive Method for Testing Peanut Seed Lots for Peanut stripe and Peanut mottle viruses by Immunocapture-Reverse Transcription-Polymerase Chain Reaction

Plant Disease ◽

10.1094/pdis.2000.84.5.559 ◽

2000 ◽

Vol 84 (5) ◽

pp. 559-561 ◽

Cited By ~ 15

Author(s):

A. G. Gillaspie ◽

R. N. Pittman ◽

D. L. Pinnow ◽

B. G. Cassidy

Keyword(s):

Polymerase Chain Reaction ◽

Reverse Transcription ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Chain Reaction ◽

Capsid Region ◽

Large Numbers ◽

Peanut Stripe Virus ◽

Pcr Method ◽

Polymerase Chain

An immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) method was developed for testing peanut (Arachis hypogaea) seed lots for infection by Peanut stripe virus (PStV) and Peanut mottle virus (PeMV). A small slice was removed from each seed distal to the radicle of a random 100-seed sample, the slices were extracted in buffer and centrifuged, and a portion of the supernatant was incubated in a tube that had been coated with antiserum to either PStV or PeMV. Following immunocapture of the virus, the tube was washed, the RT-PCR mix (with primers designed from conserved sequences within the capsid region of each virus) was placed in the same tubes, and the test completed. Results obtained on 15 previously untested seed lots from the collection indicated good correlation between virus detected by the IC-RT-PCR method and virus detected from the same seed lots by enzyme-linked immunosorbent assay (ELISA). The IC-RT-PCR method detected three lots infected with PeMV and none with PStV from 106 seed lots grown in Ecuador (results confirmed by ELISA). The IC-RT-PCR method is more sensitive than ELISA (currently used on samples consisting of five seeds), is useful for testing large numbers of seed lots of peanut germ plasm, and could be adapted to test other plants and detect other viruses.

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METHOD FOR FMD VIRUS 146S COMPONENT CONCENTRATION DETERMINATION WITH REAL-TIME REVERSE TRANSCRIPTION – POLYMERASE CHAIN REACTION IN VACCINE RAW MATERIALS

Veterinary Science Today ◽

10.29326/2304-196x-2018-2-25-26-30 ◽

2018 ◽

pp. 26-30 ◽

Cited By ~ 1

Author(s):

M. I. Doronin ◽

A. M. Timina ◽

D. A. Lozovoy ◽

V. A. Starikov ◽

D. V. Mikhalishin ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Regression Model ◽

Real Time ◽

Reverse Transcription ◽

Raw Materials ◽

Rt Pcr ◽

Chain Reaction ◽

Pcr Method ◽

Polymerase Chain

Method for determination of foot-and-mouth disease (FMD) virus 146S component concentration with real-time reverse transcription – polymerase chain reaction (rtRT-PCR) in vaccine raw materials is developed. Negative regression model of С146S = (30.269 – Ct )/4.155 type allowing determination of FMDV 146S particle concentrations based on the amplification threshold cycle values (Ct ) is proposed. It has been experimentally proven that the quantity of the 146S component determined by the real-time RT-PCR method using developed regression model and contained in the inoculation dose of FMD vaccine confers protection to the vaccinated animals against generalized FMD of A, O, Asia-1 types. rtRT-PCR method is highly sensitive and allows rapid and highly reliable estimation of the 146S antigen concentration in FMD vaccine. The method for 146S particle quantity determination by real-time RT-PCR using the regression model is reliable and demonstrates high correlation (95.5–99.0%) with the complement fixation test results.

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1504: Molecular Diagnosis and Staging of Prostate Cancer Using Clusterin in Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR)

The Journal of Urology ◽

10.1016/s0022-5347(18)33708-x ◽

2006 ◽

Vol 175 (4S) ◽

pp. 485-486

Author(s):

Sabarinath B. Nair ◽

Christodoulos Pipinikas ◽

Roger Kirby ◽

Nick Carter ◽

Christiane Fenske

Keyword(s):

Prostate Cancer ◽

Polymerase Chain Reaction ◽

Real Time ◽

Molecular Diagnosis ◽

Reverse Transcription ◽

Rt Pcr ◽

Chain Reaction ◽

Polymerase Chain

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Expression of hyaluronan synthase-3 in porcine oviducal epithelium during oestrus

Reproduction Fertility and Development ◽

10.1071/rd02100 ◽

2003 ◽

Vol 15 (2) ◽

pp. 99 ◽

Cited By ~ 22

Author(s):

Paisan Tienthai ◽

Naoko Kimura ◽

Paraskevi Heldin ◽

Eimei Sato ◽

Heriberto Rodriguez-Martinez

Keyword(s):

Polymerase Chain Reaction ◽

Reverse Transcription ◽

Hyaluronan Synthase ◽

Critical Periods ◽

Rt Pcr ◽

Chain Reaction ◽

Mean Values ◽

Polymerase Chain ◽

Sperm Reservoir ◽

Oviduct Fluid

Hyaluronan (HA) has been related to fertilization and embryo development in the pig. Furthermore, HA is present in pig oviduct fluid and the lining epithelium, particularly of the pre-ovulatory sperm reservoir. Because the mechanisms that regulate HA synthesis have not yet been clarified, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was conducted to assess the expression of mRNAs of two HA-synthesizing enzymes (has2 and has3) in the oviduct epithelium (uterotubal junction, isthmus, ampullary–isthmic junction and ampulla segments) of non-inseminated (control) and inseminated (treatment) sows at pre-, peri- and post-ovulatory oestrus. Only has3 mRNA was detected; it was present in all tubal segments of both control and treatment samples. The level of has3 expression did not vary significantly between non-inseminated and inseminated specimens, but there was a tendency (NS) for increased mean values during the peri- and post-ovulatory stages compared with pre-ovulation. It is concluded that has3 is expressed by the porcine endosalpinx epithelium and the levels of expression do not vary during the critical periods of sperm transport and fertilization, despite fluctuating levels of HA in the tubal fluid at corresponding periods.

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