Primer composition, kit and method of use for detection of expression level of key genes regulated by circadian rhythm in Drosophila melanogaster

Abstract

The invention discloses a primer composition, a kit and a use method for detecting the expression level of key genes regulated by circadian rhythm in Drosophila melanogaster. The primer composition includes amplification primers based on circadian rhythm regulation key genes and RNA internal reference. The kit includes DEPC water, 5×RT buffer, reverse transcription primer, reverse transcriptase, Z solution, 10×PCR buffer, PCR primer, 25mM magnesium chloride solution, DNA polymerase and positive control. The present invention can simultaneously detect the key genes regulating the circadian rhythm of Drosophila melanogaster, and can complete the detection of 192 samples within one day, which not only saves production and detection costs, but also improves detection efficiency; the RNA internal reference provides a control internal reference for the integrity of the sample RNA , to ensure the judgment of sample quality during the inspection process, avoid false negatives, and make the detection have better sensitivity and specificity, thereby avoiding the problem of low specificity of other detection methods.

Description

technical field [0001] The invention relates to a multiple gene detection kit and an application thereof, in particular to a detection kit for the expression level of key genes regulating circadian rhythm in Drosophila melanogaster and its application. Background technique [0002] The circadian rhythm is the law of the biological clock, which is an internal periodic rhythm with a cycle of 24 hours from day to night, which coincides with the rotation of the earth once. In order to adapt to this cyclical change in the circadian environment, organisms on the earth, including humans, have formed a series of regular regulatory mechanisms for genes, protein molecules, and signaling pathways to coordinate the circadian rhythms of various tissues and organs. Both plants and animals are regulated by circadian rhythms at all levels of life activities such as development, behavior, physiology, and metabolism. In recent years, scientists have discovered a series of important circadian...

AI Technical Summary

Problems solved by technology

The advantages of fluorescent quantitative PCR technology: high sensitivity and accurate quantification, but there are also the following disadvantages: 1) low throughput: if only one fluorescent marker is used, only one gene can be detected at a time

When a sample needs to detect multiple genes at the same time, it must be tested one by one, the cost is relatively high, the efficiency is low, the cycle is long, and it is impossible to start with a large number of samples

2) The cost is relatively high: if multiple genes need to be detected at the same time, multiple fluorescent markers are required; at present, a maximum of 4 to 5 fluorescent markers are used in common to mark each gene segment to be detected, so multiple fluorescent markers are used relatively high cost

[0006] (2) As a relatively new technology, RNA sequencing has the advantages of high sensitivity and high throughput, but it is expensive

It has the advantages of high throughput, but the technology is expensive and complicated, and the synthesis and fixation of probes are complicated, and it cannot be accurately quantified, with poor repeatability and low sensitivity.

[0008] (4) The fluorescent in situ hybridization method is to hybridize fluorescently labeled gene probes to intracellular nucleic acids in situ, and detect the brightness and quantitative gene expression levels through a fluorescent microscope, which has the disadvantages of low throughput, low sensitivity, and high cost.

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