Reverse transcription loop‐mediated isothermal amplification (RT‐LAMP), a light for mammalian transcript analysis in low‐input laboratories

Abstract

Transcript analysis is usually performed by costly, time-consuming, and expertise intensive methods, like real time-PCR, microarray, etc. However, they are not much feasible in low-input laboratories. Therefore, we implemented the reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a means of mammalian transcript analysis. Particularly, RT-LAMP was developed for buffalo aromatase cytochrome P450 (CYP19) transcript, to study its expression in 3D-cultured buffalo granulosa cells, which were exposed to lipopolysaccharide (LPS). The CYP19-RT-LAMP assay rapidly identified the LPS-induced downregulation of the CYP19 gene within 30 min at 63°C in a water bath. The assay was visualized via unaided eye by observing the change in turbidity and fluorescence, which were decreased by increasing the LPS exposure time to granulosa cells. Overall, the developed CYP19-RT-LAMP assay provided a hope on the application of RT-LAMP for mammalian transcript analysis in low-input laboratories.

Effect of Vedic music on steroidogenic gene expression in 3D‐cultured buffalo granulosa cell spheroids model system, a pilot study

“…In the present study, we did not observe any morphological difference in the shape and size of the spheroids between music treatment and control (Figure 1a,b). The typical spheroidal organization of the buffalo granulosa cells was similar to that of observed in our previous studies (Pandey et al, 2018; Yadav et al, 2018). The absence of the morphological difference between control and the treatment was also pronounced by a similar gene expression of the PCNA , a marker for cell proliferation, between control and music treatment.…”

“…Hence, there is a high probability that exposure to classic Vedic chants may influence the functions of the cultured cells. Therefore, we exposed 3D cultured buffalo granulosa cell spheroids, which have been established in our laboratory for mimicking preovulatory follicular stage (Pandey, Singh, & Onteru, 2018; Yadav, Agrawal, Pandey, Singh, & Onteru, 2018), to a classic Vedic chanting and studied the expression dynamics of steroidogenic genes in the present study.…”

Effect of dioxins in milk on 3D-cultured primary buffalo granulosa cells, a pilot study for a prospective RT-LAMP colour reaction for dioxin toxicity

“…The present study is the first study to use AHR gene expression for milk dioxin detection through LAMP reaction, as the amplification products can be detected visually with the naked eye in normal white light. The RT-LAMP was standardized for the amplification of the AHR gene from granulosa cells spheroids by standardizing the concentration of template and primers at different temperatures and incubation time according to Pandey et al (2018). On the basis of colour detection with 120 nM Hydroxynaphthol blue (HNB) dye, all the RT-LAMP positive samples of the AHR gene from the granulosa cell spheroids of both the hanging drop and polyHEMA culture systems appeared blue in colour, whereas the non-amplified and non-template controls remained in violet colour (Figure 4 and Figure 5).…”

“…Therefore, the present work was focused on granulosa cells, considering them as the best representative of the reproductive toxicity by dioxins. In our previous study, we had developed CYP19-RT-LAMP assay for mammalian tanscript analysis in low-input laboratories (Pandey, et al , 2018) by using 3D cultured buffalo granulose spheroids treated with lipopolysaccharide (LPS). It is evident by several earlier studies that the LAMP technique was more specific and sensitive without the influence of other non-targeted nucleic acids and PCR inhibitors present in the tested samples, like blood and food materials.…”

Transcriptome analysis of buffalo granulosa cells in three dimensional culture systems

“…This model has been characterized by a few key GC transcript markers and estradiol production (Yadav et al, 2018). We utilized this 3D culture model system to investigate the effect of lipopolysaccharide (LPS) on CYP19 gene expression in buffalo GC (Pandey et al, 2018), and observed that LPS caused the downregulation in CYP19 transcript in in vitro as in in vivo conditions (Pandey et al, 2018). However, a comprehensive comparative transcriptome profile is not available among freshly isolated, 2D and 3D cultured buffalo GCs to understand the gene expression patterns in the in vitro culture systems.…”