Quantitative analysis of specific mRNA species in minute cell samples by RT-PCR and flow cytometry

2001 ◽  
Vol 249 (1-2) ◽  
pp. 223-233 ◽  
Author(s):  
Veronika Stemme ◽  
Lars Rymo ◽  
Bo Risberg ◽  
Sten Stemme
Keyword(s):  
Flow Cytometry ◽  
Quantitative Analysis ◽  
Rt Pcr ◽  
Mrna Species ◽  
Specific Mrna

scholarly journals Quantitative Analysis of Fluorescence Detection Using a Smartphone Camera for a PCR Chip

Sensors ◽  
2021 ◽  
Vol 21 (11) ◽  
pp. 3917
Author(s):  
Jong-Dae Kim ◽  
Chan-Young Park ◽  
Yu-Seop Kim ◽  
Ji-Soo Hwang
Keyword(s):  
Quantitative Analysis ◽  
Fluorescence Detection ◽  
High Performance ◽  
Low Cost ◽  
Dna Amplification ◽  
Fluorescent Detection ◽  
Rt Pcr ◽  
Commercial System ◽  
Intensity Changes ◽  
The Difference

Most existing commercial real-time polymerase chain reaction (RT-PCR) instruments are bulky because they contain expensive fluorescent detection sensors or complex optical structures. In this paper, we propose an RT-PCR system using a camera module for smartphones that is an ultra small, high-performance and low-cost sensor for fluorescence detection. The proposed system provides stable DNA amplification. A quantitative analysis of fluorescence intensity changes shows the camera’s performance compared with that of commercial instruments. Changes in the performance between the experiments and the sets were also observed based on the threshold cycle values in a commercial RT-PCR system. The overall difference in the measured threshold cycles between the commercial system and the proposed camera was only 0.76 cycles, verifying the performance of the proposed system. The set calibration even reduced the difference to 0.41 cycles, which was less than the experimental variation in the commercial system, and there was no difference in performance.

scholarly journals Characterization and Use of an Antibody Detecting the CBFβ-SMMHC Fusion Protein in inv(16)/t(16;16)-Associated Acute Myeloid Leukemias

Blood ◽  
1998 ◽  
Vol 91 (6) ◽  
pp. 1882-1890
Author(s):  
David S. Viswanatha ◽  
I.-Ming Chen ◽  
Pu Paul Liu ◽  
Marilyn L. Slovak ◽  
Cathy Rankin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Fusion Protein ◽  
Fusion Gene ◽  
Type A ◽  
Detection Methods ◽  
Rt Pcr ◽  
Cytogenetic Abnormalities ◽  
Permeabilized Cells ◽  
Flow Cytometric ◽  
Acute Myeloid

The inv(16)(p13q22) and t(16;16)(p13;q22) cytogenetic abnormalities occur commonly in acute myeloid leukemia (AML), typically associated with French-American-British (FAB) AML-M4Eo subtype. Reverse transcriptase-polymerase chain reaction (RT-PCR) techniques have been recently developed to detect the presence of several variants of the resultant CBFB-MYH11 fusion gene that encodes a CBFβ-smooth muscle myosin heavy chain (SMMHC) fusion protein. We have now determined the clinical use of a polyclonal antibody [anti-inv(16) Ab] directed against a junctional epitope of the most common type of CBFβ-SMMHC fusion protein (type A), which is present in 90% of inv(16)/t(16;16) AML cases. Using flow cytometry, reproducible methods were developed for detection of CBFβ-SMMHC proteins in permeabilized cells; flow cytometric results were then correlated with cytogenetics and RT-PCR detection methods. In an analysis of 42 leukemia cases with various cytogenetic abnormalities and several normal controls, the anti-inv(16) Ab specifically detected all 23 cases that were cytogenetically positive for inv(16) or t(16;16), including a single AML case that was RT-PCR–negative. In addition to detecting all type A fusions, the anti-inv(16) Ab also unexpectedly identified the type C and type D CBFβ-SMMHC fusion proteins. Molecular characterization of one RT-PCR–positive and Ab-positive t(16;16) case with a non-type A product showed a novel previously unreported CBFB-MYH11 fusion (CBFB nt 455-MYH11 nt 1893). Flow cytometric results were analyzed using the Kolmogorov-Smirnov statistic D-value and the median value for positive samples was 0.65 (range, 0.35 to 0.77) versus 0.07 (range, −0.21 to 0.18) in the negative group (P < .0001). The overall concordance between cytogenetics and RT-PCR was 97%, whereas the concordance between flow cytometry and cytogenetics was 100%. Thus, using the anti-inv(16) Ab, all cytogenetically positive and RT-PCR–positive AML cases with inv(16) or t(16;16) could be rapidly identified. This study demonstrates the use of this antibody as an investigational tool in inv(16)/t(16;16) AML and suggests that the development of such reagents may have potential clinical diagnostic use.

scholarly journals Changes in Expression of Specific mRNA Transcripts after Single- or Re-Irradiation in Mouse Testes

Genes ◽  
2022 ◽  
Vol 13 (1) ◽  
pp. 151
Author(s):  
Kenta Nagahori ◽  
Ning Qu ◽  
Miyuki Kuramasu ◽  
Yuki Ogawa ◽  
Daisuke Kiyoshima ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Germ Cell ◽  
Sertoli Cell ◽  
Alkylating Agents ◽  
Mrna Species ◽  
Germ Cell Differentiation ◽  
Targeted Treatments ◽  
Mrna Transcripts ◽  
Specific Mrna

Alkylating agents and irradiation induce testicular damage, which results in prolonged azoospermia. Even very low doses of radiation can significantly impair testis function. However, re-irradiation is an effective strategy for locally targeted treatments and the pain response and has seen important advances in the field of radiation oncology. At present, little is known about the relationship between the harmful effects and accumulated dose of irradiation derived from continuous low-dose radiation exposure. In this study, we examined the levels of mRNA transcripts encoding markers of 13 markers of germ cell differentiation and 28 Sertoli cell-specific products in single- and re-irradiated mice. Our results demonstrated that re-irradiation induced significantly decreased testicular weights with a significant decrease in germ cell differentiation mRNA species (Spo11, Tnp1, Gfra1, Oct4, Sycp3, Ddx4, Boll, Crem, Prm1, and Acrosin). In the 13 Sertoli cell-specific mRNA species decreased upon irradiation, six mRNA species (Claudin-11,Espn, Fshr, GATA1, Inhbb, and Wt1) showed significant differences between single- and re-irradiation. At the same time, different decreases in Sertoli cell-specific mRNA species were found in single-irradiation (Aqp8, Clu, Cst12, and Wnt5a) and re-irradiation (Tjp1, occludin,ZO-1, and ZO-2) mice. These results indicate that long-term aspermatogenesis may differ after single- and re-irradiated treatment.

scholarly journals The effect of exposure of SO2 in high concentrations on CD19+ cells in reactive airway dysfunction syndrome in rat

2018 ◽  
Vol 16 ◽  
pp. 205873921879190
Author(s):  
Zhiyuan Zhang ◽  
Zhuang Ma ◽  
Wenwu Sun ◽  
Debin Ma ◽  
Jianping Cao
Keyword(s):  
Flow Cytometry ◽  
Bronchoalveolar Lavage Fluid ◽  
Lavage Fluid ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Protein Levels ◽  
Reactive Airway ◽  
Reactive Airway Dysfunction Syndrome ◽  
High Concentrations ◽  
Cd19 Expression

Reactive airway dysfunction syndrome (RADS) has a clinical manifestation similar to asthma, but some features are different between both the diseases. To probe the effect of CD19+ cells in RADS pathogenesis by inhalation of sulfur dioxide (SO2), rats were exposed to SO2 at 600 ppm for 2 h per day for 7 days and the CD19 expression in lung tissue was detected both at mRNA and protein levels by RT-PCR and western blot. The percentages of CD19+ and CD19+ CD23+ cells were measured by flow cytometry. IgG, IgA, and IgE in serum and bronchoalveolar lavage fluid (BALF) were detected by enzyme-linked immunosorbent assay (ELISA). Histological analysis was performed. The results showed that expression of CD19 in SO2 exposure group was lower than that in the control both at mRNA and protein levels ( P < 0.05). Flow cytometry analysis showed that the percentages of CD19+ and CD19+ CD23+ were significantly lower in the SO2 exposed group than that in the control ( P < 0.05). There was no difference between the control and SO2 exposed groups in both serum and BALF levels of IgG, IgA, and IgE. Pathological changes, such as chronic bronchitis, local alveolar hemorrhage, and lymphocytes infiltration were observed in SO2 exposed. RADS is a non-immunogenicity, chronic airway inflammatory disease caused by irritation of harmful factor and manifests as airway hyperresposiveness.

Expression and intracellular localization of Nanos2-homologue protein in primordial germ cells and spermatogonial stem cells

Zygote ◽  
2019 ◽  
Vol 27 (02) ◽  
pp. 82-88 ◽  
Author(s):  
Vivek Pandey ◽  
Anima Tripathi ◽  
Pawan K. Dubey
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Germ Cells ◽  
Expression Patterns ◽  
Intracellular Localization ◽  
Primordial Germ Cells ◽  
Type A ◽  
Spermatogonial Stem Cells ◽  
Single Type ◽  
Rt Pcr

SummaryThe decision by germ cells to differentiate and undergo either oogenesis or spermatogenesis takes place during embryonic development and Nanos plays an important role in this process. The present study was designed to investigate the expression patterns in rat of Nanos2-homologue protein in primordial germ cells (PGCs) over different embryonic developmental days as well as in spermatogonial stem cells (SSCs). Embryos from three different embryonic days (E8.5, E10.5, E11.5) and SSCs were isolated and used to detect Nanos2-homologue protein using immunocytochemistry, western blotting, reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. Interestingly, Nanos2 expression was detected in PGCs at day E11.5 onwards and up to colonization of PGCs in the genital ridge of fetal gonads. No Nanos2 expression was found in PGCs during early embryonic days (E8.5 and 10.5). Furthermore, immunohistochemical and immunofluorescence data revealed that Nanos2 expression was restricted within a subpopulation of undifferentiated spermatogonia (As, single type A SSCs and Apr, paired type A SSCs). The same results were confirmed by our western blot and RT-PCR data, as Nanos2 protein and transcripts were detected only in PGCs from day E11.5 and in undifferentiated spermatogonia (As and Apr). Furthermore, Nanos2-positive cells were also immunodetected and sorted using flow cytometry from the THY1-positive SSCs population, and this strengthened the idea that these cells are stem cells. Our findings suggested that stage-specific expression of Nanos2 occurred on different embryonic developmental days, while during the postnatal period Nanos2 expression is restricted to As and Apr SSCs.

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