1 Extraction of sample RNA
1 The frozen cells were frozen and allowed to stand at room temperature for 5 minutes to completely dissolve.
2 Two-phase separation 0.2 ml of chloroform was added to each 1 ml of the TRIZOL reagent-cracked sample, and the tube cap was tightly capped. After vigorously shaking the tube for 15 seconds, incubate at 15 to 30 ° C for 2 to 3 minutes. Centrifuge at 12000 rpm for 15 minutes at 4 °C. After centrifugation, the mixed liquid is divided into a lower red phenol chloroform phase, an intermediate layer and a colorless aqueous phase upper layer. The RNA is all distributed in the aqueous phase. The volume of the upper layer of the aqueous phase is approximately 60% of the TRIZOL reagent added during homogenization.
3 RNA precipitation Transfer the upper layer of the aqueous phase to a clean RNase-free centrifuge tube. An equal volume of isopropanol was added to precipitate the RNA therein, and after mixing for 10 minutes at 15 to 30 ° C, it was centrifuged at 12,000 rpm for 10 minutes at 4 ° C. At this point, the RNA precipitate that is not visible before centrifugation will form a gelatinous precipitate on the bottom and side walls of the tube.
4 RNA washing Remove the supernatant, add at least 1 ml of 75% ethanol (75% ethanol in DEPCH2O) per 1 ml of TRIZOL reagent lysed sample, and wash the RNA pellet. After mixing, it was centrifuged at 7000 rpm for 5 minutes at 4 °C.
5 RNA Drying Carefully aspirate most of the ethanol solution and allow the RNA pellet to dry in air at room temperature for 5-10 minutes.
6 Dissolving RNA Precipitate When dissolving RNA, first add 40 μl of RNase-free water and repeatedly blow it several times with a gun to completely dissolve it. The obtained RNA solution was stored at -80 ° C until use.
2 RNA quality testing
1) Determination by ultraviolet absorption method
The spectrophotometer is first zeroed with the TE solution for dilution. Then, a small amount of the RNA solution was diluted with TE (1:100), and the absorption values ​​at 260 nm and 280 nm of the spectrophotometer were read, and the concentration and purity of the RNA solution were measured.
1 concentration determination
A reading of 1 for A260 indicates 40 μg RNA/ml. The sample RNA concentration (?g/ml) is calculated as: A260 × dilution factor × 40 μg / ml. The specific calculation is as follows:
RNA was dissolved in 40 μl DEPC water, taken 5 ul, diluted 1:100 to 495 μl TE, measured A260 = 0.21
RNA concentration = 0.21 × 100 × 40 μg / ml = 840 μg / ml or 0.84 ? g / ? l
After taking 5 ul for measurement, the remaining sample RNA is 35 μl, and the total amount of remaining RNA is:
35 ?l × 0.84 ?g/?l = 29.4 ?g
2 purity detection
The ratio of A260/A280 of the RNA solution is the purity of the RNA, with a ratio ranging from 1.8 to 2.1.
2) Determination of denaturing agarose gel electrophoresis
1 made of glue
1 g of agarose was dissolved in 72 ml of water, cooled to 60 ° C, 10 ml of 10 x MOPS running buffer and 18 ml of 37% formaldehyde solution (12.3 M).
10×MOPS running buffer
Concentration component
0.4M MOPS, pH 7.0
0.1M sodium acetate
0.01M EDTA
Fill the gel plate and reserve at least 25 μl of solution for the sample well. After gelation, remove the comb, place the gel plate in the electrophoresis tank, and add a sufficient amount of 1×MOPS running buffer to cover the rubber surface for several millimeters.
2 Prepare RNA samples
Take 3?gRNA, add 3 times the volume of formaldehyde loading solution, add EB to the formaldehyde loading solution to a final concentration of 10?g/ml. The sample was denatured by incubating for 15 minutes by heating to 70 °C.
3 electrophoresis
The gel should be pre-electrophoresed for 5 min before loading, and then the sample is added to the well. 2h at 5-6V/cm voltage, electrophoresis to the bromophenol blue indicator into the gel at least 2-3cm.
4 UV transmission light observation and photographing
The bands of 28S and 18S ribosomal RNA are very bright and dense (the size depends on the type of species used to extract the RNA), and the density of the upper band is about twice that of the next band. It is also possible to observe a smaller, slightly diffuse band consisting of low molecular weight RNA (tRNA and 5S ribosomal RNA). A diffuse EB staining material can be seen between the 18S and 28S ribosomal bands, possibly composed of mRNA and other heterologous RNA. If DNA contamination occurs during RNA preparation, it will appear on the 28S ribosomal RNA band, ie, a higher molecular weight diffuse migration material or band, and the degradation of RNA appears as a dispersion of the ribosomal RNA band. The electrophoresis results were taken with a digital camera.
3 sample cDNA synthesis
1 reaction system
Serial number of reactants
1 reverse transcription buffer 2μl
2 upstream primer 0.2μl
3 downstream primer 0.2μl
4 dNTP 0.1μl
5 reverse transcriptase MMLV 0.5μl
6 DEPC water 5μl
7 RNA template 2μl
8 total volume 10μl
The solution was mixed at the bottom of the flick tube and briefly centrifuged at 6000 rpm.
2 The mixture was dried in a dry bath at 70 ° C for 3 minutes before the addition of the reverse transcriptase MMLV. Immediately after the removal, the temperature was the same in the ice water bath to the inside and outside of the tube, and then 0.5 μl of reverse transcriptase was added, and the mixture was incubated at 37 ° C for 60 minutes.
3 Immediately after taking out, dry bath at 95 ° C for 3 minutes to obtain a final solution of reverse transcription, which is a cDNA solution, and stored at -80 ° C until use.
4 Gradient dilution of standards and housekeeping genes (β-actin) real-time quantitative PCR
The concentration of the positive template for the 1β-actin positive template was 1011. Before the reaction, 3 μl was diluted 10 times (27 μl of water and mixed well) to 1010, and then diluted to 109, 108, 107, 106, 105, 104. Take a backup.
2 The reaction system is as follows:
Standard reaction system
Serial number of reactants
1 SYBR Green 1 dye 10μl
2 positive template upstream primer F 0.5μl
3 positive template downstream primer R 0.5μl
4 dNTP 0.5μl
5 Taq enzyme 1μl
6 positive template DNA 5μl
7 ddH2O 32.5μl
8 total volume 50μl
The solution was mixed at the bottom of the flick tube and briefly centrifuged at 6000 rpm.
Housekeeping gene reaction system:
Serial number of reactants
1 SYBR Green 1 dye 10μl
2 internal reference upstream primer F 0.5μl
3 internal reference downstream primer R 0.5μl
4 dNTP 0.5μl
5 Taq enzyme 1μl
6 sample sample cDNA 5μl
7 ddH2O 32.5μl
8 total volume 50μl
The solution was mixed at the bottom of the flick tube and briefly centrifuged at 6000 rpm.
3 The prepared positive standard and the test sample were simultaneously put on the machine, and the reaction conditions were: 93 ° C for 2 minutes, then 93 ° C for 1 minute, and 55 ° C for 2 minutes for a total of 40 cycles.
5 Prepare a DNA template for plotting a gradient dilution standard curve
1 For each gene to be measured, select a cDNA template that defines the gene for PCR reaction.
reaction system:
Serial number of reactants
1 10× PCR buffer 2.5 ul
2 MgCl2 solution 1.5 ul
3 upstream primer F 0.5 ul
4 downstream primer R 0.5 ul
5 dNTP mixture 3 ul
6 Taq polymerase 1 ul
7 cDNA 1 ul
8 add water to a total volume of 25ul
The solution was mixed at the bottom of the flick tube and briefly centrifuged at 6000 rpm.
35 PCR cycles (1 minute at 94 ° C; 1 minute at 55 ° C; 1 minute at 72 ° C); extended at 72 ° C for 5 minutes.
2 PCR product and DNA Ladder were electrophoresed on 2% agarose gel and stained with ethidium bromide to detect whether the PCR product was a single specific amplification band.
3 The PCR product was subjected to 10-fold serial dilution: The PCR product was subjected to 10-fold serial dilution: The PCR product concentration was set to 1 × 1010, and successively diluted to several concentration gradients of 109, 108, 107, 106, 105, and 104.
6 Real-time quantitative PCR of the test gene to be tested
1 All cDNA samples were configured with real-time quantitative PCR reaction systems.
The system configuration is as follows:
Serial number of reactants
1 SYBR Green 1 dye 10 ul
2 upstream primer 1ul
3 downstream primer 1ul
4 dNTP 1ul
5 Taq polymerase 2ul
6 sample sample cDNA 5ul
7 ddH2O 30ul
8 total volume 50 ul
The solution was mixed at the bottom of the flick tube and briefly centrifuged at 6000 rpm.
2 The prepared PCR reaction solution was placed on a Realtime PCR machine for PCR amplification reaction. The reaction conditions were: pre-denaturation at 93 ° C for 2 minutes, followed by 1 minute at 93 ° C, 1 minute at 55 ° C, 1 minute at 72 ° C for a total of 40 cycles, and finally extended at 72 ° C for 7 minutes.
7 Real-time quantitative PCR using primer lists
Primer design software: Primer Premier 5.0, and follow the following principles: primers and template sequences are closely complementary; avoid formation of stable dimer or hairpin structure between primers and primers; primers do not initiate DNA polymerization at non-target sites of the template (ie mismatch).
8 Electrophoresis
The target gene and the housekeeping gene of each sample were subjected to a Realtime PCR reaction, respectively. The PCR product was electrophoresed on a 2% agarose gel and stained with GoldView® to detect whether the PCR product was a single specific amplification band.
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