1. Extraction of RNA (see RNA extraction and reverse transcription for details)
RNA extraction from different tissue samples is suitable for different extraction methods. Because Real-TimePCR requires high quality of RNA samples, it is necessary to select an extraction method suitable for your own sample before the formal experiment, and to prevent RNA degradation during the experiment. , to maintain the integrity of the RNA.
In the extraction process of total RNA, take care to avoid the cleavage of mRNA; take 2ug for the detection of RNA by formaldehyde denaturing gel electrophoresis. If there is DNA contamination, digest it with DNaseI (because RNA is easily degraded during the treatment, it is recommended in the system). Add the appropriate amount of RNase inhibitor).
2. DNAseI digests the DNA in the sample RNA
Digesting DNA with DNaseI
Component Plus Template (RNA) 10ug
RNaseInhibitor4ul
DNaseIbuffer10ul
DNaseI10ul
DEPC treatment of H2O to 100ul
Mix, 37 ° C 90 min
3. Preparation of 1.1% agarose gel electrophoresis gel by RNA agarose gel electrophoresis:
1) Weigh 0.45 g of agarose into a flask, add 4.5 ml of 10 x MOPS buffer and 39.5 ml of DEPC water, and dissolve in a microwave oven.
2) When cooling to about 60 degrees Celsius, add 1ml of formaldehyde and shake well (to avoid air bubbles). Pour into the gel plate and solidify for 30 min.
2. Take 4 μl of each RNA sample, add 2 μl of 6×RNA electrophoresis loading buffer, and add to the denatured gel sample well.
Electrophoresis was carried out for 25 min at a voltage of 3.120V. Observed with a gel UV analyzer and photographed.
4. The results of RNA electrophoresis are shown in the figure below. Two bright bands of 28S and 18S can be seen without DNA band contamination.
Real-time PCR (Real-TimePCR) experimental procedure IV. RNA reverse transcription to cDNA
Reverse transcription program (take MBI's M-MLV as an example)
Component addition (20ul system) addition (40ul system)
Template (RNA) 0.1~2.5ug (adjusted according to the brightness of the strip) 3ug (adjusted according to the brightness of the strip)
Primer T18 (50uM) (or other primer) 2.0ul4.0ul
DEPC treatment of H2O to 12.5ul to 25ul
Mix well, 70 ° C for 5 min, immediately ice bath 5 * buffer 4.0 ul ul
dNTP (10mM) 2.0ul4.0ul
RNaseInhibitor0.5ul1.0ul
Mix, 37 ° C 5 min
M-MLV1.0ul2.0ul
42 ° C 60 min, 70 ° C 10 min
Selection of Reverse Transcription Primers and Requirements for Real-Time PCR Primer Design 1) Random hexamer primers:
When a particular mRNA is difficult to copy its full length sequence by containing a sequence that terminates the reverse transcriptase, a non-specific primer, a random hexamer primer, can be used to copy the full length mRNA. In this way, all RNA molecules in the system act as cDNA* strand templates, and PCR primers confer the required specificity during amplification. 96% of the cDNA normally synthesized with this primer is derived from rRNA.
2) Oligo (dT):
It is a method that is specific only to mRNA. Since most eukaryotic mRNAs have a 3'-end Poly(A+) tail, this primer is paired with it and only mRNA can be transcribed. Since Poly(A+) RNA accounts for only 1-4% of total RNA, cDNA synthesized by such primers is smaller in size and complexity than random hexamers as primers and cDNA obtained. It is especially suitable for detecting the expression of multiple genes, which can save reverse transcription reagents. cDNA can be used multiple times. It can be used to detect the expression of rare genes and quantitatively detect the expression level of specific mRNA from a very small number of cells.
3) Specific primers:
The zui-specific reverse transcription method uses an oligonucleotide containing the complementary sequence of the target RNA as a primer. If the PCR reaction uses two specific primers, the synthesis of the * strand can be initiated by a paired primer close to the 3' end of the mRNA. . The use of such primers produces only the desired cDNA, resulting in more specific PCR amplification.
When performing RealTimePCR, the pair of primers used in the SYBRGreenI/EvaGreen method and the primers for general PCR have different parameters required for primer design. Primer design requirements:
1Tm=55-65°C
2GC=30-80%
3 PCR amplification product length: the product size of the primer is not too large, generally between 80-300 bp.
4 The annealing temperature of the primer is high, generally above 60 °C.
Particular care should be taken to avoid the presence of primer dimers and non-specific amplification. Moreover, the primer design should take into account the ability of the primer to be immune to genomic DNA contamination, ie, the primer should cross the exon, and the primer is the primer region that can cross the exon, so that it can be more effectively free from genomic DNA contamination. Impact.
As for the design software, PRIMER3, PRIMER5, PRIMEREXPRESS should be ok. The key to doing dye method is to find suitable primers and prevent pollution. For primers, you have to be prepared to pick out one or two primers that can be used from a large number of primers - finding the right primer is not easy.
5. cDNA and primer quality detection Take 0.2ml thin-wall PCR tube, numbered. Dye 2×PCRTaqMix10 ul was added to each tube; 0.5 ul each of the forward and reverse primers (primer concentration 10 uM) was added, and 1 ul of the mixed cDNA was added to the tube. Add water to each tube to 20 ul.
Component addition amount 2 × PCRTaqMix10ul
10uMPrimerFW0.5ul
10uMPrimerRV0.5ul
TemplateDNA1ul
Mix ddH2O to 20ul
Mix and place in TP600PCR instrument. 95 ° C 5 min; 95 ° C 15 s, 60 ° C 35 s, 40 cycles; 72 ° C 5 min; 4 ° C pause.
8 μl of each amplification product was taken, and the DL2000 molecular weight standard was 5 μl/lane. Electrophoresis was carried out for 25 min at a voltage of 120 V on a 1% agarose gel. Observed with a gel UV analyzer.
Primers with good specificity and high amplification efficiency were selected as primers for real-time fluorescence.
6. Using a relatively quantitative method to analyze the expression of the target gene:
Due to different objective factors such as different yields after RNA purification and different efficiency of RNA reverse transcription to cDNA, the initial sample concentration used for quantitative analysis is different. Therefore, in the study of gene expression regulation, some housekeeping genes are used for standardization. Correct the difference due to the initial concentration of the sample. Commonly used housekeeping genes are beta-actin, GAPDH, 18SrRNA, and the like. Therefore, at least two genes, a target gene and a housekeeping gene are required for gene expression regulation analysis.
Seven, quantitative PCR detection:
Take 0.2 ml thin wall PCR tubes and number them separately. 2×qPCRTaqMix12.5 ul was added to each tube, and 10 μM of the gene forward and reverse primer mixture was 0.5 ul, and the corresponding cDNA was 1 ul each. No template was used as a negative control in one tube. Add water to each tube to 25 ul.
Component addition template (cDNA) / ddH2O1.0ul
10uM primer F/R0.5ul
2×qPCRMix12.5ul
ddH2O11.0ul
Mix and place in a SLAN fluorescence quantitative PCR machine. After pre-denaturation at 95 ° C for 5 min, 95 ° C 15 s → 65 ° C 35 s (fluorescence detection), 40 cycles. Fluorescence quantitative PCR generally sets the annealing and amplification to a temperature, and only considers the gradient when there is a problem with the amplification.
8. Amplification curve and dissolution curve Real-time PCR (Real-Time PCR) experimental procedure Real-time PCR (Real-Time PCR) experimental procedure The dissolution curve is all single peak indicating specific amplification.
In general, the fluorescence amplification curve can be divided into three phases: a fluorescent background signal phase, a fluorescent signal exponential amplification phase, and a plateau phase, the shape of which is a smooth S-shaped curve.
If there are many inflection points in the fluorescent background signal stage, the possible reason is that the system is not mixed or there is solid impurities;
If the probe is quickly lifted up and then down the probe, the possible reason is that the amount of the template in the system is too high. It is recommended to use the template after dilution.
If the primer dimer is present, the negative control will appear to be head-up, which is difficult to avoid in Real-TimePCR;
If the dissolution curve of the negative control appears to be the same peak as in the sample, indicating that there is contamination in the system configuration, the experimental results are not available.
There are three possibilities for double peaks in the dissolution curve:
1 primer peak, the primer peak is usually the first of the two peaks, the elimination method is to reduce the amount of primers in the system or redesign the primers;
2 When the difference in gene expression is made, the amplified peak of DNA is likely to occur (only when the primer crosses the intron). The reason is that there is DNA contamination when extracting RNA, which can be verified by electrophoresis. At this time, the RNA sample should be re-digested. DNA
3 Amplification is non-specific, then re-expand amplification conditions or redesign and verify primers.
9. Calculation method for expression difference Absolute quantification calculates the copy number of the starting template through the standard curve; the relative quantification method compares the expression of the treated sample with the untreated sample target transcript or the target transcript at different phases. Differences in expression between differences.
The 2-△△CT method is a simple method for analyzing the relative changes in gene expression in real-time quantitative PCR experiments.
In some cases, it is not necessary to quantify the transcripts, only the relative gene expression differences need to be given. Obviously, we say that the X gene is 2.5 times more expressed after some treatment than the expression of the gene is increased from 1000 copies/cell to 2500 copies/cell.
Derivation of 2-△△CT method (see real-time quantitative PCR and 2-△△CT method for analysis of relative expression of genes)
Supplement: After DNaseI digests the DNA in the sample RNA, the sample needs to be re-extracted with chloroform. The specific experimental procedure is as follows:
After digestion, the total volume is 10Oul, the volume is too small, which is not conducive to extraction. We often add 200ul of DEPC water.
1. Add an equal volume of chloroform (about 300 ul) to the centrifuge tube, shake it vigorously, and mix well until a white flaky precipitate appears in the middle layer. Centrifuge at 14000 rpm for 8 min at 4 ° C, and take the supernatant (about 250 ul).
2. Add 1/10 volume of NaAC (3M) (about 25 ul) and pre-cooled equal volume of isopropanol (about 280 ul) and let stand at -20 °C for 20 min.
Centrifuge at 3.4 ° C for 14,000 rpm for 15 min, remove the supernatant, taking care not to touch the precipitate.
4. Add 1 ml of 75% ethanol, centrifuge at 14000 rpm for 3 min at 4 ° C, and remove the supernatant.
5. Centrifuge instantaneously, carefully aspirate the residual liquid at the bottom of the centrifuge tube with a 200 ul (or 10 ul) tip (do not absorb the sediment at the bottom of the tube).
6. Place the tube on the ultra-clean table and let it dry for about 5-10 minutes (do not dry completely, otherwise it will be difficult to dissolve). Add 30~50μl of RNase-free water, vortex for 1min, shake for 30sec, and centrifuge instantaneously.
7. The extracted RNA is immediately subjected to downstream experiments or stored at -20 °C.
RNA extraction from different tissue samples is suitable for different extraction methods. Because Real-TimePCR requires high quality of RNA samples, it is necessary to select an extraction method suitable for your own sample before the formal experiment, and to prevent RNA degradation during the experiment. , to maintain the integrity of the RNA.
In the extraction process of total RNA, take care to avoid the cleavage of mRNA; take 2ug for the detection of RNA by formaldehyde denaturing gel electrophoresis. If there is DNA contamination, digest it with DNaseI (because RNA is easily degraded during the treatment, it is recommended in the system). Add the appropriate amount of RNase inhibitor).
2. DNAseI digests the DNA in the sample RNA
Digesting DNA with DNaseI
Component Plus Template (RNA) 10ug
RNaseInhibitor4ul
DNaseIbuffer10ul
DNaseI10ul
DEPC treatment of H2O to 100ul
Mix, 37 ° C 90 min
3. Preparation of 1.1% agarose gel electrophoresis gel by RNA agarose gel electrophoresis:
1) Weigh 0.45 g of agarose into a flask, add 4.5 ml of 10 x MOPS buffer and 39.5 ml of DEPC water, and dissolve in a microwave oven.
2) When cooling to about 60 degrees Celsius, add 1ml of formaldehyde and shake well (to avoid air bubbles). Pour into the gel plate and solidify for 30 min.
2. Take 4 μl of each RNA sample, add 2 μl of 6×RNA electrophoresis loading buffer, and add to the denatured gel sample well.
Electrophoresis was carried out for 25 min at a voltage of 3.120V. Observed with a gel UV analyzer and photographed.
4. The results of RNA electrophoresis are shown in the figure below. Two bright bands of 28S and 18S can be seen without DNA band contamination.
Real-time PCR (Real-TimePCR) experimental procedure IV. RNA reverse transcription to cDNA
Reverse transcription program (take MBI's M-MLV as an example)
Component addition (20ul system) addition (40ul system)
Template (RNA) 0.1~2.5ug (adjusted according to the brightness of the strip) 3ug (adjusted according to the brightness of the strip)
Primer T18 (50uM) (or other primer) 2.0ul4.0ul
DEPC treatment of H2O to 12.5ul to 25ul
Mix well, 70 ° C for 5 min, immediately ice bath 5 * buffer 4.0 ul ul
dNTP (10mM) 2.0ul4.0ul
RNaseInhibitor0.5ul1.0ul
Mix, 37 ° C 5 min
M-MLV1.0ul2.0ul
42 ° C 60 min, 70 ° C 10 min
Selection of Reverse Transcription Primers and Requirements for Real-Time PCR Primer Design 1) Random hexamer primers:
When a particular mRNA is difficult to copy its full length sequence by containing a sequence that terminates the reverse transcriptase, a non-specific primer, a random hexamer primer, can be used to copy the full length mRNA. In this way, all RNA molecules in the system act as cDNA* strand templates, and PCR primers confer the required specificity during amplification. 96% of the cDNA normally synthesized with this primer is derived from rRNA.
2) Oligo (dT):
It is a method that is specific only to mRNA. Since most eukaryotic mRNAs have a 3'-end Poly(A+) tail, this primer is paired with it and only mRNA can be transcribed. Since Poly(A+) RNA accounts for only 1-4% of total RNA, cDNA synthesized by such primers is smaller in size and complexity than random hexamers as primers and cDNA obtained. It is especially suitable for detecting the expression of multiple genes, which can save reverse transcription reagents. cDNA can be used multiple times. It can be used to detect the expression of rare genes and quantitatively detect the expression level of specific mRNA from a very small number of cells.
3) Specific primers:
The zui-specific reverse transcription method uses an oligonucleotide containing the complementary sequence of the target RNA as a primer. If the PCR reaction uses two specific primers, the synthesis of the * strand can be initiated by a paired primer close to the 3' end of the mRNA. . The use of such primers produces only the desired cDNA, resulting in more specific PCR amplification.
When performing RealTimePCR, the pair of primers used in the SYBRGreenI/EvaGreen method and the primers for general PCR have different parameters required for primer design. Primer design requirements:
1Tm=55-65°C
2GC=30-80%
3 PCR amplification product length: the product size of the primer is not too large, generally between 80-300 bp.
4 The annealing temperature of the primer is high, generally above 60 °C.
Particular care should be taken to avoid the presence of primer dimers and non-specific amplification. Moreover, the primer design should take into account the ability of the primer to be immune to genomic DNA contamination, ie, the primer should cross the exon, and the primer is the primer region that can cross the exon, so that it can be more effectively free from genomic DNA contamination. Impact.
As for the design software, PRIMER3, PRIMER5, PRIMEREXPRESS should be ok. The key to doing dye method is to find suitable primers and prevent pollution. For primers, you have to be prepared to pick out one or two primers that can be used from a large number of primers - finding the right primer is not easy.
5. cDNA and primer quality detection Take 0.2ml thin-wall PCR tube, numbered. Dye 2×PCRTaqMix10 ul was added to each tube; 0.5 ul each of the forward and reverse primers (primer concentration 10 uM) was added, and 1 ul of the mixed cDNA was added to the tube. Add water to each tube to 20 ul.
Component addition amount 2 × PCRTaqMix10ul
10uMPrimerFW0.5ul
10uMPrimerRV0.5ul
TemplateDNA1ul
Mix ddH2O to 20ul
Mix and place in TP600PCR instrument. 95 ° C 5 min; 95 ° C 15 s, 60 ° C 35 s, 40 cycles; 72 ° C 5 min; 4 ° C pause.
8 μl of each amplification product was taken, and the DL2000 molecular weight standard was 5 μl/lane. Electrophoresis was carried out for 25 min at a voltage of 120 V on a 1% agarose gel. Observed with a gel UV analyzer.
Primers with good specificity and high amplification efficiency were selected as primers for real-time fluorescence.
6. Using a relatively quantitative method to analyze the expression of the target gene:
Due to different objective factors such as different yields after RNA purification and different efficiency of RNA reverse transcription to cDNA, the initial sample concentration used for quantitative analysis is different. Therefore, in the study of gene expression regulation, some housekeeping genes are used for standardization. Correct the difference due to the initial concentration of the sample. Commonly used housekeeping genes are beta-actin, GAPDH, 18SrRNA, and the like. Therefore, at least two genes, a target gene and a housekeeping gene are required for gene expression regulation analysis.
Seven, quantitative PCR detection:
Take 0.2 ml thin wall PCR tubes and number them separately. 2×qPCRTaqMix12.5 ul was added to each tube, and 10 μM of the gene forward and reverse primer mixture was 0.5 ul, and the corresponding cDNA was 1 ul each. No template was used as a negative control in one tube. Add water to each tube to 25 ul.
Component addition template (cDNA) / ddH2O1.0ul
10uM primer F/R0.5ul
2×qPCRMix12.5ul
ddH2O11.0ul
Mix and place in a SLAN fluorescence quantitative PCR machine. After pre-denaturation at 95 ° C for 5 min, 95 ° C 15 s → 65 ° C 35 s (fluorescence detection), 40 cycles. Fluorescence quantitative PCR generally sets the annealing and amplification to a temperature, and only considers the gradient when there is a problem with the amplification.
8. Amplification curve and dissolution curve Real-time PCR (Real-Time PCR) experimental procedure Real-time PCR (Real-Time PCR) experimental procedure The dissolution curve is all single peak indicating specific amplification.
In general, the fluorescence amplification curve can be divided into three phases: a fluorescent background signal phase, a fluorescent signal exponential amplification phase, and a plateau phase, the shape of which is a smooth S-shaped curve.
If there are many inflection points in the fluorescent background signal stage, the possible reason is that the system is not mixed or there is solid impurities;
If the probe is quickly lifted up and then down the probe, the possible reason is that the amount of the template in the system is too high. It is recommended to use the template after dilution.
If the primer dimer is present, the negative control will appear to be head-up, which is difficult to avoid in Real-TimePCR;
If the dissolution curve of the negative control appears to be the same peak as in the sample, indicating that there is contamination in the system configuration, the experimental results are not available.
There are three possibilities for double peaks in the dissolution curve:
1 primer peak, the primer peak is usually the first of the two peaks, the elimination method is to reduce the amount of primers in the system or redesign the primers;
2 When the difference in gene expression is made, the amplified peak of DNA is likely to occur (only when the primer crosses the intron). The reason is that there is DNA contamination when extracting RNA, which can be verified by electrophoresis. At this time, the RNA sample should be re-digested. DNA
3 Amplification is non-specific, then re-expand amplification conditions or redesign and verify primers.
9. Calculation method for expression difference Absolute quantification calculates the copy number of the starting template through the standard curve; the relative quantification method compares the expression of the treated sample with the untreated sample target transcript or the target transcript at different phases. Differences in expression between differences.
The 2-△△CT method is a simple method for analyzing the relative changes in gene expression in real-time quantitative PCR experiments.
In some cases, it is not necessary to quantify the transcripts, only the relative gene expression differences need to be given. Obviously, we say that the X gene is 2.5 times more expressed after some treatment than the expression of the gene is increased from 1000 copies/cell to 2500 copies/cell.
Derivation of 2-△△CT method (see real-time quantitative PCR and 2-△△CT method for analysis of relative expression of genes)
Supplement: After DNaseI digests the DNA in the sample RNA, the sample needs to be re-extracted with chloroform. The specific experimental procedure is as follows:
After digestion, the total volume is 10Oul, the volume is too small, which is not conducive to extraction. We often add 200ul of DEPC water.
1. Add an equal volume of chloroform (about 300 ul) to the centrifuge tube, shake it vigorously, and mix well until a white flaky precipitate appears in the middle layer. Centrifuge at 14000 rpm for 8 min at 4 ° C, and take the supernatant (about 250 ul).
2. Add 1/10 volume of NaAC (3M) (about 25 ul) and pre-cooled equal volume of isopropanol (about 280 ul) and let stand at -20 °C for 20 min.
Centrifuge at 3.4 ° C for 14,000 rpm for 15 min, remove the supernatant, taking care not to touch the precipitate.
4. Add 1 ml of 75% ethanol, centrifuge at 14000 rpm for 3 min at 4 ° C, and remove the supernatant.
5. Centrifuge instantaneously, carefully aspirate the residual liquid at the bottom of the centrifuge tube with a 200 ul (or 10 ul) tip (do not absorb the sediment at the bottom of the tube).
6. Place the tube on the ultra-clean table and let it dry for about 5-10 minutes (do not dry completely, otherwise it will be difficult to dissolve). Add 30~50μl of RNase-free water, vortex for 1min, shake for 30sec, and centrifuge instantaneously.
7. The extracted RNA is immediately subjected to downstream experiments or stored at -20 °C.
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