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

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...

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Bibliographic Details
Published in:Journal of cellular biochemistry 2018-06, Vol.119 (6), p.4334-4338
Main Authors: Pandey, Mamta, Singh, Dheer, Onteru, Suneel K.
Format: Article
Language:English
Subjects:
Aromatase
Assaying
buffalo
Cost analysis
CYP19
Cytochrome
Cytochrome P450
DNA microarrays
Fluorescence
Granulosa cells
Laboratories
Lipopolysaccharides
LPS
Mammals
Reverse transcription
RT‐LAMP
Turbidity
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Summary: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. The developed RT‐LAMP assay for buffalo CYP19 gene provided a hope to implement RT‐LAMP for mammalian transcript analysis in low‐input laboratories.
ISSN:0730-2312
1097-4644
DOI:10.1002/jcb.26624