SYBR Green I is a fluorescent dye with a green excitation wavelength that binds to all regions of the dsDNA double helix groove and binds to all double-stranded DNA molecules of all sequences. In the free state, SYBR Green I fluoresces weakly, but once bound to double-stranded DNA and embedded in dsDNA, the fluorescence is greatly enhanced.
Therefore, the fluorescence signal intensity of SYBR Green I is related to the amount of double-stranded DNA, and the fluorescence signal intensity of SYBR Green I varies with the amount of dsDNA in different periods of PCR amplification. The amount of double-stranded DNA present in the PCR system can be detected based on the fluorescence signal, and can be calculated and analyzed based on the control.
Before starting the experiment, prepare the reagents and consumables needed for the experiment.
Reagents include: specific PCR primers, freshly extracted total RNA.
The following kits are used: Transcriptor FirstStrand cDNA Synthesis Kit for cDNA first-strand synthesis, which contains all of the experimental components required for the cDNA reaction as well as the relevant positive control reaction components. control reaction.
The LightCycler? 480 SYBR Green I Master Kit for LightCycler? 480 contains all the components required for PCR, such as hot-start Taq DNA Polymerase and reaction buffer, dNTP mix, SYBR Green I dye and MgCl2 formal test in tube 1. Prior to the start of the formal test, thaw each reagent and kit component on ice and place on ice for use. Note: SYBR Green I Master needs to be protected from light.
Instruments and consumables required: Roche LightCycler? 480 fully automated real-time quantitative PCR instrument and accompanying 96- or 384-well plates, transparent sealing film and other disposable consumables. Thermo Scientific Arktik PCR instrument, single-channel pipettes, Nunc iceboxes, and QSP cartridge tips from Thermo Fisher Scientific.
Next, we entered the experimental part, the procedure of this experiment is: firstly, we synthesized the first strand of cDNA by reverse transcription of freshly extracted RNA, and then we performed real-time fluorescence quantitative PCR, which includes: the configuration of the SYBR Green reaction system, the setup of the PCR program, the PCR experiments, the editing of the samples, and the analysis of the results.
The first step is to synthesize the first strand of cDNA by reverse transcription:
Note: Wear gloves for all RNA-related operations to prevent RNase contamination. Follow the instructions of the kit to perform the relevant operations.
The Template primer mix was configured according to the system recipe in Rnase free sterilized PCR tubes on ice in a total system of 13 μl. This experiment is a combination of anchored oligo dT primer and random hexamer primer for reverse transcription. The NTC control was first configured by adding 10 μl of water from tube 9, 1 μl of 50 mM oligo dT primer from tube 6, and 2 μl of 0.6 mM random hexamer primer from tube 5, and mixing well. Then the system of sample tube 1 was configured by adding 1 μl of total RNA, 1 μl of oligo dT primer, 2 μl of random hexamer primer, and finally 9 μl of water to a total volume of 13 μl, and mix well. For the other tubes, refer to the configuration of tube 1 and add the reaction system sequentially. Note: The amount of template can be adjusted to 10ng-5μg of total RNA and 1-100ng of mRNA.
If the concentration of RNA sample is low, 10μg/ml of MS2 RNA can be added to stabilize the template RNA.
Denaturation of the reaction mix at 65℃ for 10min in the PCR instrument can effectively reduce the secondary structure of the RNA. The secondary structure of RNA can be effectively reduced. After heating, the reaction mix was rapidly chilled on ice for 5 min, and the following reagents were added to the reaction Template primer mix in the order of the system recipe: 5× reverse transcriptase buffer in tube 2, dNTP mix in tube 4, 40 U/μl of RNase inhibitor in tube 3, and 20 U/μl of reverse transcriptase in tube 1, in a final volume of 20 μl.< /p>
If the sample is to be stabilized, the reaction mix was prepared in the PCR instrument at 65°C for 10 min. /p>
If the number of samples is large, configure the reaction mix before dispensing and mix by pipetting carefully without vortexing. After mixing, centrifuge briefly in an instantaneous centrifuge to bring the sample and reaction solution to the bottom of the tube. Place the tube in the PCR and set up the program according to the primers used and the fragment length of the target mRNA. In this experiment, the reaction temperature and time were set as follows: 25℃ for 10min, 55℃ for 30min, and after the reaction was completed, the reverse transcriptase was inactivated by heating at 85℃ for 5min, and then the reaction was stopped by placing it on ice. The reaction product can be stored at 2-8℃ for 1-2h, or at -15 to -25℃ for a longer period of time.
The cDNA product can be used in subsequent PCR reactions without purification. A 20-μl PCR system yields 2-5 μl of cDNA product for amplification. The RNase H-activated reverse transcriptase in this kit removes the RNA template after cDNA synthesis, minimizing its impact on subsequent PCR.
For this part of the experiment, we used Roche's SYBR Green I Master kit for basic absolute quantification.
For this experiment***, we set up 5 standard samples, including a blank control labeled 0, and 5 reverse transcription samples, including an NTC control. Due to the large number of samples, we prepared the total system without template, and then divided it into 10 tubes, and added the templates of each sample, and then divided each sample into 3 duplicate wells.
Add 2×Master Mix with green cap, 10x Primer upstream and downstream primers, and PCR grade water according to the system recipe. After the total system was prepared, the system was pipetted gently and then divided into 10 tubes of 55μl each, and the cDNA concentration of each sample was added to each of the 10 tubes. 6μl of water was added to STD zero instead of the template, and the remaining STD was added with 6μl of the standard template that had been diluted step by step. For the reverse transcription samples, add 6 μl of the adjusted concentration of template DNA and mix well with NTC, then dispense 20 μl of each sample per well into a 96-well plate. Cover the 96-well plate with a sealing film and place the plate in a suitable centrifuge at 1500 x g for 2 min. Place the prepared 96-well plate in the LightCycler?
Double-click to open the 1.5 software of LightCycler? 480 and log in, automatically enter the software interface, click new experiment, select SYBR Green I mode in the Detection Format option, click OK to complete the setting of the detection channel, then set up the reaction volume, for 96 modules, the reaction system is between 10μl-100μl. For the 96 module, the reaction system is between 10μl-100μl, and in this experiment, the reaction system is 20μl.
In Program Name, enter the reaction name pre incubation, set 1 cycle for pre-denaturation, no need to collect fluorescence, set the execution temperature and time to 95℃ for 10min, the view will be adjusted in real time according to the setting.
Click the Add button, enter Amplification, define the number of amplification cycles as 45, and select Quantification for fluorescence collection, then set the temperature and time of the PCR amplification cycle to 95℃ for 10s, click the Add button, set the annealing temperature and time to 60℃ for 10s, and select Acquisition Mode by default for both steps. Acquisition Mode is selected None by default, continue to click the Add button, set the extension temperature and time to 72℃ 20s, Acquisition Mode is selected Single, Ramp Rate is adjusted according to the automatic value, no need to set again.
To set the dissolution curve, enter Melting Curves in the new line of Program, 1 cycle number, select Melting Curves for Analysis Mode, set the time and temperature to 95℃ for 5s, click the Add button, add the new set temperature and time to 65℃ for 1min, the above two steps. Acquisition Mode is selected as None by default, click Add button to set the temperature to 95℃, Acquisition Mode is selected as Continuous, and no other settings need to be changed.
Set the process of holding Cooling, only 1 cycle, no need to collect fluorescence, set the temperature and time for 40 ℃, 1min. after the completion of the setup, click on the right side of the Save button to save the setup program. At this point, the whole PCR cycle system and temperature settings have been set.
Click Start run to start the PCR reaction, and then you can monitor the amplification of the samples in real time on the software.
At the end of the experiment, click Sample Editor to enter the sample editing area.
Select Abs Quant in Select Workflow and edit the sample according to how it is laid out in the plate. Set up negative controls, blanks, standards, and unknown samples.
In Select Sample, check the sample wells you want to set up. Enter the name of the selected sample group in Sample Name in the next column, and select the type of sample group in Sample Type, and finally click Make Replicates to set up duplicate wells. When you set up the standard, you need to fill in the number of copies, just fill in the number of dilutions and the initial concentration. After editing the sample, you can analyze the data. If necessary, you can also edit the subset.
After editing the samples, you can analyze the results.
Click the sum button at the bottom of the left column, and all the information of the experiment will appear accordingly, including: the designed reaction program, the analysis of the experimental results, etc.
Click the sum button at the bottom of the left column.
Clicking on analysis will allow you to analyze the sample in detail and accurately based on the data available.
Click Abs Quant second derivative max to bring up the create new analysis window, select the distribution of the analyzed samples in the Subset option, and click OK to bring up the analysis graph: the amplification curve of the corresponding samples.
Standard Curve is the standard curve based on the standards. The left side of the curve is labeled with amplification efficiency, slope, intercept, linear relationship, and error rate. Generally, the smaller the "Error" value, the more accurate the experiment.
The closer the amplification efficiency is to "2", the better the amplification reaction.
In the data table, the Cp values for individual samples are displayed, along with the corresponding sample concentration values.
Data statistics by replicate wells show the Cp mean, variance, concentration mean, and variance.