CN112342300B - Fluorescence quantitative detection reference gene of 7 tissues of red crayfish as well as screening method and application thereof - Google Patents
Fluorescence quantitative detection reference gene of 7 tissues of red crayfish as well as screening method and application thereof Download PDFInfo
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Abstract
A fluorescence quantitative detection reference gene for 7 tissues of red swamp crayfish and a screening method and application thereof belong to the technical field of molecular biology. The invention comprises the following steps: 7, internal reference genes GAPDH and GST for tissue fluorescence quantitative detection of the red crayfish; the special primer for amplifying the reference gene comprises a nucleotide sequence of an upstream primer F1 for amplifying the GAPDH gene as shown in SEQ ID No.1, a nucleotide sequence of a downstream primer R1 as shown in SEQ ID No.2, a nucleotide sequence of an upstream primer F2 for amplifying the GST gene as shown in SEQ ID No.3, and a nucleotide sequence of a downstream primer R2 as shown in SEQ ID No.4. The invention screens out proper and stable reference genes for the research of the gene expression of different tissues of the red swamp crayfish, is beneficial to accurately analyzing the gene expression rule of the red swamp crayfish in experiments with different purposes and provides necessary molecular basis for the subsequent research of the molecular mechanism of the red swamp crayfish.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a reference gene for fluorescence quantitative detection of 7 tissues of red swamp crayfish, a screening method and application thereof.
Background
Reference genes (Reference genes), whose expression is stable in various tissues, at different growth stages or developmental stages, are often used as references in detecting gene expression levels, and their selection can directly affect the accuracy of the qRT-PCR results. Studies have shown that most of the reference genes are stably expressed only under specific tissue conditions and are not applicable to all species. Therefore, strict screening of reference genes is necessary for different biological experiments. Red crayfish (Cherax quadricarinatus), also known as quadricarinatus, is native to australia and is also known as australian crayfish. With the development of the molecular mechanism of the phenomena of growth, shelling and the like of the red swamp crayfish, quantitative expression research on related genes of the crayfish under various background conditions is required, and therefore, screening of a reference gene which can be stably expressed in different tissues of the red swamp crayfish is very important.
Disclosure of Invention
Aiming at the defects in the prior art, the application aims to design and provide the reference gene for quantitatively detecting the tissue fluorescence of the red crayfish 7 and the screening method and the application thereof.
The reference gene for tissue fluorescence quantitative detection of the red swamp crayfish 7 is characterized in that the reference gene comprises a GAPDH gene (GenBank accession number: KM 538172) and a GST gene (GenBank accession number: JF 284517.1).
The special primer for amplifying the internal reference gene for the quantitative fluorescence detection of the tissues of the red crayfish 7 as claimed in claim 1 is characterized in that the nucleotide sequence of an upstream primer F1 for amplifying the GAPDH gene is shown as SEQ ID NO.1, and the nucleotide sequence of a downstream primer R1 is shown as SEQ ID NO. 2; the nucleotide sequence of the upstream primer F2 for amplifying the GST gene is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream primer R2 is shown as SEQ ID NO.4.
The reference gene is applied to the fluorescence quantitative PCR of different tissues of the red crayfish.
The special primer is applied to fluorescence quantitative PCR of the red crayfish.
The screening method of the reference gene for the fluorescence quantitative detection of the 7 tissues of the red crayfish is characterized by comprising the following steps of:
(1) Collecting a sample: randomly selecting 5 adult female and male red swamp crayfishes respectively, cleaning, taking 7 tissues of muscles, hepatopancreas, intestines, ovaries, spermary, gills and eyes, respectively placing the tissues in sterilized EP tubes, quickly freezing by liquid nitrogen, and transferring to a refrigerator at the temperature of-80 ℃ for freezing and storing;
(2) Respectively extracting RNA of each tissue obtained in the step (1) through cryopreservation, and preserving at-80 ℃;
(3) Carrying out reverse transcription on the RNA obtained in the step (2) by using a reverse transcription kit to obtain cDNA;
(4) Designing a primer: selecting GAPDH, beta-ACTIN, AK, HSP90, naK, cq-CL, alpha-TUB, 18S rRNA, GST and Rps18 genes as candidate reference genes, respectively designing and synthesizing fluorescent quantitative F and R primers, wherein the length of the primers is between 100 and 250 bases, and the annealing temperature is 60 ℃;
(5) And (3) primer screening: the cDNA of each tissue of the red swamp crayfish is mixed in equal proportion, diluted into 5 concentration gradients according to the proportion of 1Increasing, the smooth arrival plateau phase according to the amplification curve, and the melting curve is unimodal R 2 > 0.99、105% >Amplification efficiency E>95% of standard screening qualified primers;
(6) And (3) carrying out qRT-PCR amplification on the cDNA of the 7 tissues of the red crayfish by using the F and R primers of the candidate internal reference genes qualified in the step (5), setting 5 biological repeats and 3 technical repeats, reading Ct values after the amplification is finished, calculating delta Ct values of the candidate internal reference genes, respectively introducing the Ct values into the GENorm, normFinder and BestKeeper software, analyzing the stability of the candidate internal reference genes, and obtaining the internal reference genes with good stability for the fluorescence quantitative detection of the 7 tissues of the red crayfish.
The screening method of the reference gene for the fluorescent quantitative detection of the tissues of the red crayfish 7 is characterized in that the step (2) is specifically operated as follows:
(a) Transferring the tissue obtained in the step (1) to an RNase-free EP tube, adding 1000ul of Trizol reagent, and grinding on ice until the tissue becomes a homogenate;
(b) Standing the homogenate at room temperature for 10min;
(c) Centrifuging at 12000 rpm at 4 deg.C for 3 min with a low temperature centrifuge, and transferring the supernatant to a new EP tube;
(d) Adding 200ul chloroform, violently shaking or vortexing until uniform, and centrifuging at 12000 rpm for 10min at 4 ℃ by a low-temperature centrifuge;
(e) Withdrawing the supernatant and repeating step (c);
(f) Extracting the supernatant in the step (e), adding 500ul of isopropanol, fully reversing and uniformly mixing, standing at-20 ℃ for 30min, and centrifuging at 12000 rpm for 10min by a low-temperature centrifuge at 4 ℃;
(g) Discarding the supernatant, washing twice with 75% ethanol solution, air drying, adding DEPC treated water, and dissolving;
(h) The concentration is measured by using NanoDrop 2000, and whether the RNA is degraded or not is judged by electrophoresis of 1% agarose gel;
(i) The undegraded RNA was stored in a-80 ℃ freezer.
The screening method of the reference gene for the 7-tissue fluorescence quantitative detection of the red swamp crayfish is characterized in that the step (3) is specifically as follows:
removing genomic DNA: after a genome DNA removing reaction system is configured, incubating the reaction system in a PCR instrument at 42 ℃ for 2min, taking out a reaction solution and storing the reaction solution at 4 ℃, wherein the genome DNA removing reaction system is 5 XgDNA Eraser Buffer 2ul, gDNA Eraser 1ul, the RNA 1ug obtained in the step (2), RNase Free ddH 2 O is complemented to 10ul system;
(II) carrying out reverse transcription: preparing a reverse transcription reaction system on ice, incubating at 37 ℃ for 15 min, inactivating at 85 ℃ for 3 s, taking out and storing at 4 ℃, wherein the reverse transcription reaction system is 5 XPrimeScript Buffer 2 4ul, primeScript RT Enzyme Mix1 1ul, RT Primer Mix 1ul, and the reaction liquid 10ul and RNase Free ddH obtained in the step (I) 2 O is complemented to 20ul.
The screening method of the reference gene for the 7-tissue fluorescence quantitative detection of the red crayfish is characterized in that the qRT-PCR amplification reaction system in the steps (5) and (6) is as follows: hieff UNICON qPCR SYBR Green Master Mix12.5ul,10uM F primer 0.4 ul,10uM R primer 0.4 ul, cDNA1ul, 10.7 ul of sterile ultrapure water; reaction conditions step1: 95. 30sec, step2 cycle 40 times: 95 ℃ 5 sec,60 ℃ 30sec.
The invention screens out proper and stable reference genes, namely the GAPDH gene and the GST gene, for the gene expression research of different tissues of the red swamp crayfish, is beneficial to accurately analyzing the gene expression rule of the red swamp crayfish in experiments with different purposes, and provides necessary molecular basis for the subsequent research of the molecular mechanism of the red swamp crayfish.
Drawings
FIG. 1 is an illustration of a standard melting curve, wherein A-J are reference candidate genes GAPDH, β -ACTIN, AK, HSP90, naK, cq-CL, α -TUB, 18S rRNA, GST and Rps18, respectively;
FIG. 2 is a standard graph of candidate reference genes GAPDH, β -ACTIN, AK, HSP90, naK, cq-CL, α -TUB, 18S rRNA, GST and Rps 18.
Detailed Description
The invention will be further explained below with reference to the drawings and examples.
The embodiment is as follows:
1. sample collection
The red chelonian used in the experiment is obtained from honghai aquaculture limited company in haining city, zhejiang province. Randomly selecting 5 adult female and male red swamp crayfishes respectively, cleaning the surface of the crayfish body by DEPC (DePC) treated water, taking 7 tissues of muscle, hepatopancreas, intestine, ovary, spermary, gill and eye by using sterile scissors and tweezers, placing the tissues in a sterilized EP (EP) tube with the volume of 1.5ml, quickly freezing the tissues in liquid nitrogen, and transferring the frozen tissues to a refrigerator with the temperature of 80 ℃ below zero for freezing and storing.
2. Extraction of RNA for reverse transcription
(1) The tissue samples stored in liquid ammonia were taken out, an appropriate amount of tissue blocks were transferred to an EP tube without rnase, 1000ul of Trizol reagent was added, and ground on ice using a Tiangen electric tissue grinder until the tissue blocks became completely homogenates.
(2) Standing the homogenate at room temperature for 10min to fully dissociate the nucleoprotein complex.
(3) Centrifugation was carried out at 12000 rpm for 3 min at 4 ℃ using a cryocentrifuge, after which the supernatant was transferred to a new EP tube using a pipette.
(4) 200ul of chloroform was added, shaken vigorously or vortexed until homogeneous, and centrifuged at 12000 rpm for 10min at 4 ℃ using a cryocentrifuge.
(5) And (4) extracting the supernatant and repeating the step (3).
(6) And (4) extracting the supernatant obtained in the step (5), adding 500ul of isopropanol, fully reversing and uniformly mixing, standing at-20 ℃ for 30min, and centrifuging at 12000 rpm for 10min at 4 ℃ by using a low-temperature centrifuge.
(7) The supernatant was discarded, washed twice with 75% ethanol solution (DEPC treated water configuration), the residual alcohol was air dried, and an appropriate amount of DEPC treated water was added to dissolve.
(8) The concentration was determined using a NanoDrop 2000 and the presence of degradation of RNA was judged by electrophoresis on a 1% agarose gel.
(9) Qualified RNA samples were stored in a-80 ℃ freezer and prepared for reverse transcription.
3. Reverse transcription
The reverse transcription experiment was performed using the PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) reverse transcription Kit from Takara, japan, and the specific steps were as follows:
(1) Removing genome DNA, incubating at 42 deg.C for 2min in PCR instrument, taking out, and storing at 4 deg.C, wherein the reaction system is shown in Table 1.
TABLE 1 reaction System for removing genomic DNA
(2) Reverse transcription, preparing reverse transcription system, and this step needs to be carried out on ice. Incubate at 37 deg.C for 15 min in PCR instrument, inactivate at 85 deg.C for 3 s, take out and store at 4 deg.C, and the reverse transcription reaction system is listed in Table 2.
TABLE 2 reverse transcription reaction System
4. Primer design and screening
GAPDH, beta-ACTIN, AK, HSP90, naK, cq-CL, alpha-TUB, 18S rRNA, GST and Rps18 genes are selected as candidate reference genes, the base sequences of the genes are inquired through an NCBI database, and https:// www.genetic script.com/tools/real-time-pcr-taqman-primer-design-tool online website is used for designing fluorescent quantitative primers (shown in table 3), wherein the design conditions are that the product length is between 100 and 250 bases, the annealing temperature is 60 ℃, and the primers are synthesized by the limited post-committee engineering (Shanghai) engineering.
TABLE 3 primer sequences of candidate reference genes of red crayfish
Using a real-time fluorescent quantitative PCR apparatus (Bio-Ra)d CFX Real-time PCR Detection System, CFX96, USA) according to Hieff UNICON ® The primers for 10 reference genes were screened for validation using the qPCR SYBR Green Master Mix (antibody method, no Rox) instructions. The cDNAs of 5 different tissues of the red crayfish were mixed in equal proportions, and then diluted to 5 concentration gradients in a ratio of 1. The 25uL reaction system is shown in Table 4.
TABLE 4 Standard Curve PCR 25ul reaction System and reaction conditions
After the reaction, a melting curve (see FIG. 1) was confirmed, which was sharp and a single peak, and the occurrence of a hetero-peak indicated that the primer specificity was poor. In terms of cDNA concentration (log) 5 ) The value is the abscissa, the Ct value is the ordinate, the standard curve is drawn, and the R of the standard curve is read 2 The value is obtained. According to the formula E = (5) (−1/slope)−1 ) The amplification efficiency (E) of each reference gene was calculated at X100%. The smooth arrival plateau phase of the amplification curve and the melting curve are unimodal R 2 > 0.99、105% >Amplification efficiency (E)>95% of standard screening qualified primers, qualified primer sequence and product fragment size, R 2 The values are given in Table 3 and the standard curve is given in FIG. 2. The qualified primers are used for the stability analysis of the internal reference genes in the next step.
5. Analysis of stability of reference Gene
The cDNAs of 7 tissues of red crayfish were amplified using primers for each gene listed in Table 3, and 5 biological replicates and 3 technical replicates were set. The qRT-PCR reaction system and reaction procedure are shown in Table 4. And after the program is finished, reading the Ct value, calculating the delta Ct value of each candidate reference gene, respectively introducing the Ct value into the software of geonorm, normFinder and BestKeeper, and analyzing the stability of each candidate reference gene. The geNorm program screens out the reference genes with better stability by calculating the M value of the stability of each reference gene, and the judgment standard is that the stability of the reference genes is better when the M value is smaller, otherwise, the stability is worse. The calculation principle of the NormFinder is similar to that of the geNorm program, and the judgment standard is that the reference gene with the minimum expression stability value is the most suitable reference gene. The BestKeeper judgment principle is that the larger the correlation coefficient is, the smaller the standard deviation and the variation coefficient is, the better the stability of the internal reference gene is, and otherwise, the worse the stability is; when SD > 1, the reference gene expression is unstable.
As is clear from Table 5, among the different tissues of the red crayfish, rps18, 18S rRNA and GST showed small changes in Ct value, and β -ACTIN showed the largest change. As shown in Table 6, GST, AK and GAPDH were more stable and β -ACTIN was the lowest in different tissues as analyzed by geNorm software; through analysis of NormFinder software, AK, GST and GAPDH in different tissues have higher stability, and beta-ACTIN has the lowest stability; the stability of Rps18, GAPDH and alpha-TUB is higher and the stability of beta-ACTIN is lowest in different tissues according to the analysis of BestKeeper software. The results are combined, and the GST and GAPDH genes have good expression stability in each tissue of the red crayfish, and are suitable for fluorescent quantitative PCR between different tissues of the red crayfish to be used as reference genes.
Table 5 raw Ct value data of reference genes
TABLE 6 results of analysis of geNorm and NormFiner for differences in expression of reference genes
Sequence listing
<110> Zhejiang province academy of agricultural sciences
<120> fluorescence quantitative detection reference gene of 7 tissues of red crayfish and screening method and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> F1(F1)
<400> 1
<210> 2
<211> 20
<212> DNA
<213> R1(R1)
<400> 2
<210> 3
<211> 20
<212> DNA
<213> F2(F2)
<400> 3
<210> 4
<211> 20
<212> DNA
<213> R2(R2)
<400> 4
Claims (1)
- The application of GST gene as internal reference gene for fluorescence quantitative PCR detection of 7 tissues of red crayfish, wherein the 7 tissues are muscle, liver pancreas, intestine, ovary, testis, gill and eye.
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