Real-time quantitative fluorescence PCR test method based on double external references of RNA and DNA and application thereof

Abstract

The invention relates to the field of bioinformatics, in particular to a real-time quantitative fluorescence PCR test method based on double external references of RNA and DNA and an application thereof, comprising the following steps: selecting external reference genes, preparing mRNA and DNA of the external reference genes, preparing premix of the mRNA and DNA of the external reference genes; adding the premix while extracting the samples, collecting the general mRNA and DNA part in the samples; carrying out real-time quantitative fluorescence PCR and calculating copy number of genes to be tested and mRNA and DNA of the external reference genes; carrying out data processing and normalization. The invention also discloses the application of the method in the technology that gene chips are used to analyze gene expression. The invention improves experimental design work on testing of genetic transcription level, which results in obtaining the true genetic transcription level of a certain gene instead of the relative level thereof, thus providing new concepts and frameworks for integration of gene expression data and gene copy data.

Description

technical field

[0001] The invention relates to the field of bioinformatics, in particular to a real-time quantitative fluorescent PCR detection method with RNA and DNA double external references and its application. Background technique

[0002] Since Crick put forward the central dogma of molecular biology in 1970, people have done a lot of research on the mechanism of gene transcription and expression and its regulation. The Northern hybridization method was the first method used to measure gene transcription. Later, RT-PCR (reverse transcription-PCR, namely: reverse transcription-polymerase chain reaction) generated by combining reverse transcription and polymerase chain reaction provided a sensitive and convenient semi-quantitative determination method. In recent years, real-time quantitative polymerase chain reaction (abbreviated as: qPCR) method has gradually become the main method for quantitative determination of gene expression due to its sensitivity, accuracy and...

Examples

Embodiment 1

[0040] 1. Treatment of experimental silkworms:

[0041] The eggs of the experimental silkworm 54A were provided by the Sericulture Research Institute of the Chinese Academy of Agricultural Sciences, and were reared with artificial feed at 25°C after being bred and hatched. The larvae were dissected on the 3rd day when they reached the 5th instar, and the middle silk gland, posterior silk gland, fat body, Malpighian duct and midgut were collected, washed with redistilled water three times, and stored at -70°C.

[0042] 2. Selection of RNA and DNA external reference:

[0043] The piggyBac transposon was found in the Lepidoptera cell line TN-368, which consists of two transposable arms and a coding sequence for the transposase IFP2. Since IFP2 does not exist in silkworm, IFP2 was selected as the external reference in this embodiment.

[0044] 3. Detect the ratio of RNA and DNA external reference:

[0045] Plasmid pPigT.7 is made by inserting T7-IFP2 expression cassette into ve...

Embodiment 2

[0108] Example 2 Detection of A3, GAPDH, and 28S rRNA expression in various tissues of the silkworm:

[0109] Use the detection method in Example 1 to A3, GAPDH, the transcription product of 28S rDNA gene and gene and external reference IFP2mRNA and DNA in silk gland of Bombyx mori middle part, posterior silk gland, fat body, Malpighian tubule and midgut extract The copy number in was determined. A3, the expression levels of GAPDH and 28SrDNA genes per copy in these tissues were calculated according to the above method. Their apparent transcript levels vary greatly among different tissues (as shown in Table 3).

[0110] Table 3 Apparent transcription levels of A3, GAPDH and 28S rRNA genes in silkworm tissues

[0111] organize

A3

GAPDH

28S rRNA

middle silk gland

(MSG)

122.25±4.83

10.794±1.728

5935.5±353.3

posterior silk gland

(PSG)

1097.6±276.4

154.39±6.72

9016.1±458.8

...

Embodiment 3

[0115] Example 3 Detection of the expression of silk fibroin gene and sericin 1 gene in silkworm:

[0116] The detection method is the same as in Example 1. The apparent transcription levels of silk fibroin heavy chain gene (FibH), light chain gene and sericin 1 gene in each tissue of silkworm were determined. The results showed that FibH and FibL were exclusively expressed in the posterior silk gland, and Ser-1 was exclusively expressed in the middle silk gland. The apparent transcription level of FibL in the posterior silk gland (192029±30739) was similar to that of Ser-1 in the middle silk gland (196539±12455), while the apparent transcription level of FibH in the posterior silk gland (106453 ±13667) is about half of FibL.

[0117] If the expression of FibH and FibL in the posterior silk gland and the expression of Ser-1 in the middle silk gland were normalized with A3, GAPDH or 28S rRNA as usual, the expression of Ser-1 in the middle silk gland was higher than that of Fi...

technical field

[0001] The invention relates to the field of bioinformatics, in particular to a real-time quantitative fluorescent PCR detection method with RNA and DNA double external references and its application. Background technique

[0002] Since Crick put forward the central dogma of molecular biology in 1970, people have done a lot of research on the mechanism of gene transcription and expression and its regulation. The Northern hybridization method was the first method used to measure gene transcription. Later, RT-PCR (reverse transcription-PCR, namely: reverse transcription-polymerase chain reaction) generated by combining reverse transcription and polymerase chain reaction provided a sensitive and convenient semi-quantitative determination method. In recent years, real-time quantitative polymerase chain reaction (abbreviated as: qPCR) method has gradually become the main method for quantitative determination of gene expression due to its sensitivity, accuracy and...

Examples

Embodiment 1

[0040] 1. Treatment of experimental silkworms:

[0041] The eggs of the experimental silkworm 54A were provided by the Sericulture Research Institute of the Chinese Academy of Agricultural Sciences, and were reared with artificial feed at 25°C after being bred and hatched. The larvae were dissected on the 3rd day when they reached the 5th instar, and the middle silk gland, posterior silk gland, fat body, Malpighian duct and midgut were collected, washed with redistilled water three times, and stored at -70°C.

[0042] 2. Selection of RNA and DNA external reference:

[0043] The piggyBac transposon was found in the Lepidoptera cell line TN-368, which consists of two transposable arms and a coding sequence for the transposase IFP2. Since IFP2 does not exist in silkworm, IFP2 was selected as the external reference in this embodiment.

[0044] 3. Detect the ratio of RNA and DNA external reference:

[0045] Plasmid pPigT.7 is made by inserting T7-IFP2 expression cassette into ve...

Embodiment 2

[0108] Example 2 Detection of A3, GAPDH, and 28S rRNA expression in various tissues of the silkworm:

[0109] Use the detection method in Example 1 to A3, GAPDH, the transcription product of 28S rDNA gene and gene and external reference IFP2mRNA and DNA in silk gland of Bombyx mori middle part, posterior silk gland, fat body, Malpighian tubule and midgut extract The copy number in was determined. A3, the expression levels of GAPDH and 28SrDNA genes per copy in these tissues were calculated according to the above method. Their apparent transcript levels vary greatly among different tissues (as shown in Table 3).

[0110] Table 3 Apparent transcription levels of A3, GAPDH and 28S rRNA genes in silkworm tissues

[0111] organize

A3

GAPDH

28S rRNA

middle silk gland

(MSG)

122.25±4.83

10.794±1.728

5935.5±353.3

posterior silk gland

(PSG)

1097.6±276.4

154.39±6.72

9016.1±458.8

...

Embodiment 3

[0115] Example 3 Detection of the expression of silk fibroin gene and sericin 1 gene in silkworm:

[0116] The detection method is the same as in Example 1. The apparent transcription levels of silk fibroin heavy chain gene (FibH), light chain gene and sericin 1 gene in each tissue of silkworm were determined. The results showed that FibH and FibL were exclusively expressed in the posterior silk gland, and Ser-1 was exclusively expressed in the middle silk gland. The apparent transcription level of FibL in the posterior silk gland (192029±30739) was similar to that of Ser-1 in the middle silk gland (196539±12455), while the apparent transcription level of FibH in the posterior silk gland (106453 ±13667) is about half of FibL.

[0117] If the expression of FibH and FibL in the posterior silk gland and the expression of Ser-1 in the middle silk gland were normalized with A3, GAPDH or 28S rRNA as usual, the expression of Ser-1 in the middle silk gland was higher than that of Fi...

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