quantitative PCR

Summary

Quantitative PCR can be used for:

(1) quantification of the amount of PCR starting template by analyzing the PCR end-product or monitoring the PCR process using external or internal reference as a standard; (2) precise quantification of the amount of starting template by monitoring the PCR process (monitoring the amplification efficiency) using external standard (fluorescent hybridization probe to ensure specificity), and at the same time, internal control to effectively exclude the false-negative results (amplification efficiency is zero).

Operation method

quantitative PCR

Principle

Quantitative PCR (Real-time Polymerase Chain Reaction, or Real-time PCR, instant PCR), also known as Quantitative real time polymerase chain reaction (Q-PCR/qPCR/rt-qPCR, quantitative instant PCR, instant quantitative PCR), is a method for detecting each polymerase chain reaction during DNA amplification using a fluorescent dye. Q-PCR/qPCR/rt-qPCR, quantitative real-time PCR, quantitative real-time PCR), is a method and technology to detect the total amount of products after each PCR cycle by fluorescent dye in the DNA amplification reaction.

Materials and Instruments

Restriction endonuclease Reverse transcriptase Heat-stable DNA polymerase dCTP Target nucleic acid
Amplification buffer dNTP storage solution Placenta RNase inhibitor
Agarose or polyacrylamide gels Shielded tips Tubes Positive displacement pipettes PCR instruments

Move

I. Materials

1. Buffers and solutions

10X amplification buffer

4 dNTP storage solution (20 mmol/L, pH 8.0)

MgCl₂ ( 50 mmol/L)

Placenta RNase inhibitor (20 units/ul)

2. Enzymes and buffers

Suitable restriction endonuclease

Reverse transcriptase (100~200 units/ul)

Heat-stable DNA polymerase

3. Radioactive material

[ ^α-32P ] dCTP (specific activity: 3000 Ci/mmol) at a concentration of 10 mCi/mI

4. Gel

Agarose gel or polyacrylamide gel

5. Nucleotides and oligonucleotides

plus a known amount of reference molecule (DNA or RNA)

Forward primer (20 umol/L) and reverse primer (20 umol/L) in water

Target nucleic acids

6. Specialized equipment

Shielded tips for automated micropipettes

Centrifuge tubes (0.5 ml, thin-walled centrifuge tubes for amplification reactions)

Forward-displacement pipettes

PCR Instrument

Methods

Preparation of reference templates

1. Apply the guidelines for the design and preparation of reference templates in the Introduction to the protocol to the design and preparation of reference templates suitable for this task. It is important to determine the concentration of the reference template as carefully as possible, preferably with a fluorophore. Another method is to estimate the amount of reference template after gel electrophoresis and ethidium bromide (EB ) staining.

2. Make a series of 10-fold dilutions of the reference template with water at concentrations ranging from ^10-6 to ^10-12 mol/L. After applying these dilutions in Step 3, the dilutions should be stored at -70 °C after use for use in Step 8. Remember to use water, not TE, as a solvent to prevent altering the concentration of magnesium ions in the PCR.

Preparation of cDNA samples

3. If the starting molecule is RNA, denature this target RNA molecule by incubating it at 75°C for 5 min, followed by accelerated cooling by inserting the sample tube into ice water. Then, a series of reverse transcription reactions are immediately set up in 0.5 ml centrifuge tubes containing a gradient of reference template. Add the following reagents to each of these series of reaction tubes:

10X Amplification Buffer 2 ul
20 mmol/L 4 dNTP Mix 1 ul
20 umol/L Antisense Primer 2.5 ul
approx. 20 units/ul Placental RNase Inhibitor 1 ul
50 mmol/L MgCl₂ 1 ul
Denatured Target RNA 10 pg~1.0 ug
100~200 units /ul Reverse transcriptase 1 ul
10-fold dilution of reference template 1 ul
_H2O Make up to 20 ul
MgCl₂ is required for the transcriptase to function.

Reaction tubes were warmed on a 37°C water bath for 60 min; then the sample tubes were heated to 95°C and left for 20 min to denature and inactivate the reverse transcriptase.

靶核酸分子与参考分子的扩增

4. 用 0.5 ml 灭菌离心管、扩增管或灭菌微量滴定板，将步骤 3 中建立的系列反应管的每一只管内依次加入如下试剂，建立扩增反应：

反转录反应（步骤 3）祥品或靶 DNA 样品 5 ul
20 umol/L 正义引物 1.5 ul
20 umol/L 反义引物 1.25 ul
10X Amplification Buffer 5 ul
[ ^α-32P ] dCTP (3000 Ci/mmol) 10 uCi
20 mmol/L 4 dNTP mixes 1 ul
Taq DNA Polymerase 2 units
_H2O make up to 50 ul

5. If the PCR instrument is not equipped with a heating cap, a drop of mineral oil (about 50 ul) should be added to the top of the reaction mix to prevent the sample from heating and cooling during the PCR reaction. If the PCR instrument is not equipped with a heating cap, a drop of mineral oil (~50 ul) should be added to the top layer of the reaction mixture to prevent evaporation of the sample during multiple heating and cooling cycles of the PCR reaction. Alternatively, if a hot-start PCR program is applied, add a drop of paraffin oil to the top of the reaction mixture. Place a centrifuge tube or microtitre plate on the PCR instrument.

6. Perform PCR amplification as follows. Typical procedures are denaturation, denaturation and polymerization (extension reactions); the corresponding cycling conditions and temperatures are listed below:




Detection and quantification of PCR products

A number of methods are available for the detection and quantification of PCR amplification products. These include solid-phase analysis, anion-exchange high-performance liquid chromatography (HPLC) and fluorescent labeling of amplified nucleic acid molecules (Reischl and Kochamvski 1995). However, these specialized techniques, which are so esoteric, are neither widely available nor strictly necessary. Accurate results can also be obtained by applying more standardized methods based on gel electrophoresis, e.g., application of fluorescently labeled primers, quantitative analysis of amplification products by fully automated DNA sequence analysis (Porcher et al. 1992), fluorimetry, and computerized gel image analysis systems in which the gel is stained with ethidium bromide or other chelating stains (Schneeberger et al. 1992). Schneeberger et al. 1995; Tsai and Wiltbank 19%), or tracking the radioactivity during PCR amplification.

7. Analysis and determination of amplification products

(1) In the case of size differences between the applied reference template molecules and the target sequences

① A 20-ul sample was taken from each reaction tube, and the size of the amplification products was analyzed by gel electrophoresis and radioautography. ① Take 20 ul of sample from each reaction tube and analyze the size of the amplification product by gel electrophoresis and autoradiography.

Recover the amplified electrophoretic bands of the reference template and the target sequence from the gel and determine the activity of each band by liquid scintillation counting only. Optionally, the electrophoresis gel can be scanned and analyzed with a suitable detector [e.g., Ambis scanner or phosphorimager]

③ Calculate the relative amount of the two radiolabeled DNAs in each PCR reaction.

(2) When a new restriction site is used or a naturally occurring site is missing from all reference templates

① At the end of the last cycle of the amplification reaction, heat the samples at 94℃ for 5 min.

② Allow the samples to cool down to room temperature, and then take 20 ul of reaction solution from each tube and digest it with an appropriate restriction endonuclease.

③ Analyze the size of the amplified fragments by gel electrophoresis and autoradiography.

④ Recover amplified electrophoretic bands of the reference template and target sequence from the gel and determine the activity of each band by liquid scintillation counter. Alternatively, the electrophoresis gel can be scanned and analyzed with a suitable detector (e.g., Ambis scanner or phosphor imager).

⑤ Calculate the relative amount of the two radiolabeled DNAs in each PCR reaction.

8. Based on the results of the assay, determine the concentration of the reference template that can be nearly equal to the amplification product of the target sequence. Set up another series of amplification reactions (see step 4) to further narrow down the concentration of the reference template. In addition to the conventional components, this series of reactions should also include: reference templates at successively increasing dilutions (1:10, 2:10, 3:10, 4:10, etc.).

9. Repeat Steps 5, 6, and 7. Determine the ratio of reference template to amplification yield of the target sequence for each amplification reaction, graph the amount of reference template added to each amplification reaction against this ratio, and identify the corresponding point on the resulting straight line (i.e., where exactly equal amounts of target sequence were obtained in the reaction). (i.e., the amount of reference template in the reaction that yields exactly the same amount of amplification product as the target sequence). Calculate the concentration of the target sequence in the initial sample.

Caveat

RT-qRCR affects the reliability of the analysis Key point (Key point):1. The analytical results depend on the quantity and quality of templates as well as the rational design of the assay.

2. Non-standardization of the reverse transcription reaction affects the stability of the test.

3. Data analysis should be highly objective, and confusing and erroneous results can be obtained from the analysis results if the analysis is not done rationally, so it is necessary to minimize the variability and maximize the reproducibility by evaluating the quality of each component of the RT-qPCR, and it is also necessary to follow a common guideline for the analysis of the data. The need for standardization of gene expression analysis is compatible with human clinical diagnostic analysis.

Common Problems

Sourced from Molecular Cloning Laboratory Guide, Third Edition" Translated by Huang Peitang et al.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/