Real time PCR mRNA quantification experiment route selection F&Q
Question 1: The relative and absolute quantitative selection of Real time PCR mRNA ;
Determination of mRNA, generally using relative quantification, because of absolute quantification, standard preparation and quantification is difficult, please refer to this document: http:// ;
“ Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 22DDCT Method â€
Kenneth J. Livak* and Thomas D. Schmittgen
Applied Biosystems, Foster City, California 94404; and †Department of Pharmaceutical Sciences, College of Pharmacy
Washington State University, Pullman, Washington 99164-6534
Question 2: The choice of dye method and probe method for Real time PCR ;
A: It is economical to use the dye method to determine the amount of mRNA expression; the dye method can obtain the product specificity by comparing the melting peak position of the melting curve; the target gene and the internal reference gene are detected in the tube;
The dye method can be selected from Real time dye PCR PreMIX, A2010A0112, A2010A0112-2, A2010A0112-3 (http://)
The probe method is mainly applied to the detection of viral RNA ; and the single point mutation ( SNP ); combined detection of multiple genes;
If an mRNA has multiple subtypes, it is difficult to find a portion of the amplification product that is identical to design primers, and the probe method can be used to detect the primers and probes in the homologous region, and the combined detection of multiple genes; However, if the dye method is used, there may be multiple products, the melting curve is not a single peak, and the detected data is difficult to read; in this case, the probe method is applied; the probe method can also be used to simultaneously detect the target gene in the same reaction. And the case of the internal reference gene; and the dye rule must be in charge of the test.
Probe method can be used: hot start Taqman-PCR PreMix kit (probe) A2010A0103 ( http:// );
Hot-Start Taqman-PCR Probe Multi-Fluorescence Premix A2010A0114 (http://)
One-step RT-Real time PCR probe method Premix, A2010A0115s, A2010A0115L (http://)
Question 3: Real time PCR relative quantification in the selection of standard curve method and deltadelta CT method ;
A: The deltadelta CT method is generally selected; when the amplification efficiency differs greatly, the standard curve method can be used for detection and calculation;
Question 4: How does the standard curve method in Real time PCR relative quantification work:
A: It is mainly the production of relatively quantitative standards. There are generally three methods:
1 , a more complicated method: plasmid
Biotnt provides specific real time PCR mRNA primers for internal reference genes ( custom primer pairs ) Â ( http:// ), real time PCR experiments on the target gene to obtain PCR amplification products; PCR amplification products were transferred into plasmids to obtain relatively quantitative standards;
2 , the general method 1 : a strong CDNA template
Biotnt provides specific real time PCR mRNA primers for internal reference genes ( custom primer pairs ) Â ( http:// ), real time PCR experiments on the target gene, select a strong CDNA template, generally CT between 20-25 , 5 times on this CDNA Gradient dilution, usually diluted four to five points; Real time PCR was performed on the gradient-diluted cDNA , the CT value was plotted on the ordinate, and the abscissa was the logarithm of the relative concentration of the cDNA template, and a standard curve was drawn.
3 , the commonly used method: PCR amplification products
If a strong CDNA template cannot be found in Method 2 , it is also possible to use the PCR product as a relative quantitative standard; the matters needing attention are: The concentration of the PCR product is high, and the product fragment of real time PCR is relatively short. It is easy to cause PCR contamination during the dilution process, so pay attention to the operation.
PCR products were typically diluted in a subsequent times, as a template 1ul was added to the system, this time CT about 10 cycles, it is recommended that a template for PCR of times, make 100-fold dilution, establishing a standard curve;
Note: The standard curve generally crosses the CT of the sample you are testing to be more accurate.
Standards should be distributed as close as possible to the CT of your sample; therefore, the dilution factor of the standard is not necessarily the same;
Question 5: Real time PCR mRNA relative quantification in the selection of internal reference ;
A: Generally, genes with relatively abundant abundance such as beta-actin , GAPDH and 18sRNA are selected as internal reference genes;
The internal reference gene can also be selected according to the reference;
Biotnt provide reference gene-specific real time PCR mRNA primer (custom primer pairs) Â ( http:// )
The strict selection of the internal reference gene should have the following steps:
1. Select some representative samples for the experimental group you want to test;
2. Unified extraction of RNA ;
3. Determine the RNA content; select the same amount of RNA for reverse transcription;
4. Identify the candidate internal reference genes, prepare primers for the internal reference genes; and obtain an optimal system;
5. determining the CT value of each candidate internal reference gene of the transcribed CDNA ;
6. Calculate the average CT , SD and CV of each candidate internal reference gene;
7. Select CV as a qualified internal reference gene;
8. If the CV of multiple candidate internal reference genes is relatively small, the effect of your experiment on the internal reference is relatively small; it is recommended to select the candidate internal reference gene that is relatively close to your target gene CT as the internal reference gene of the experiment.
Question 6: Real time RT-PCR uses a two-step method or a one-step method for reverse transcription ;
answer:
If you want to test multiple genes of interest at the same time, it is generally recommended to use a two-step method, first using the mRNA CDNA First-Strand Synthesis Kit (http://) to obtain the CDNA; Then use Biotnt fluorescent quantitative PCR Premix kit ( fluorescent dye ) ( http:// ) and specific real time PCR mRNA primers ( custom primer pairs ) Â ( http:// ), simultaneous detection of multiple genes, high-throughput and high-efficiency experiments; simultaneous detection of multiple genes; up to 96- well plates Perform double-repeat detection of 48 genes;
If the gene you are testing is particularly low, or if the sample is difficult to extract, or if the primer is particularly difficult to design, you can use the Real time RT-PCR one - step protocol; that is, the mRNA you extracted, using a one-step RT-Real time PCR Dye method Premix A2010A0116L (http://), and specific real time PCR mRNA primers ( custom primer pairs ) Â ( http:// ), a relative quantitative study of mRNA expression by real time PCR ; because of the use of specific primers for reverse transcription, increased detection specificity, elimination Interference with other mRNAs .
Question 7: Real time RT-PCR reverse transcription primers, using oligodT or random primers, or specific primer selection ;
Generally detecting mRNA , OligodT is usually used as a reverse transcription primer; for reverse transcription of mRNA , the specificity is relatively high;
Specific primers can be used for mRNAs with specific sequences known, or other RNAs to be detected (eg, viruses, etc.) with the highest specificity;
Random primers can be used for reverse transcription of total RNA of unknown sequence with the lowest specificity;
Question 8: What is the principle of Biotnt RNA extraction?
A: Biotnt 's RNA extraction is based on the Trizol method; ( http:// ). Is a quick and convenient Trizol total RNA extraction reagent, total RNA was extracted from a sample can be animal tissues, cells or the like and a variety of microorganisms. It is the method commonly used in laboratories today. The RNA used in the reverse transcription and real time PCR experiments of Biotnt late did not require additional purification.
You can also register as a Biotnt user, ask us questions, we will choose a more general question listed on the website.
Mc Trader Brand
Guangdong Jia Mei Biological Technology Co.Ltd , https://www.jaymeimask.com