TRzol LS (Liquid Sample RNA Extraction Reagent)

Product attributes:

Storage conditions:

TRzol LS can be stored stably for 12 months at room temperature. However, for optimal results, we recommend storing in an environment of 2-8 ° C.

Important Notice:

Toxic substances that come into contact with the skin or are accidentally ingested can cause burns. Once in contact with the skin, immediately wash with a large amount of detergent and clean water. If you feel uncomfortable, see a doctor and seek the correct treatment plan with other ingredients.

Product Introduction

TRzol LS reagent is a reagent that directly extracts total RNA from cells or tissues. It can maintain the integrity of RNA when breaking and dissolving cells. After adding chloroform and centrifuging, the sample was divided into a water sample layer, an intermediate layer, and an organic layer. RNA exists in the aqueous layer. After collecting the water sample layer above, RNA can be recovered by isopropanol precipitation. This method has good separation effects on small and large amounts of tissues and cells, whether they are humans, animals, plants, or bacteria. The simplicity of TRzol LS reagent operation allows for the same

Process multiple samples simultaneously. All operations can be completed within one hour. The total RNA extracted by TRzol can avoid DNA and protein contamination and can be used for RNA blotting analysis, spot hybridization, poly (A)+screening, in vitro translation, RNAse protection analysis, and molecular cloning. If used for PCR, it is recommended to use amplification grade DNase I to process the extracted total RNA when two primers are located within a single exon.

Notes:

1. Isolate RNA samples from a small amount of tissue (1-10mg) or cells (102-104 cells): Add 800 µ l TRzol to the tissue or cells. After the sample is lysed, add chloroform and perform the extraction operation in step 2. Before precipitating RNA with isopropanol, add 5-10 μ g of RNAase free glycogen as a carrier for the water sample layer. To reduce its viscosity, aspirate twice with a No. 26 syringe before adding chloroform to cut off genomic DNA. Glycogen will remain in the water sample layer and co elute with RNA. It will not inhibit the synthesis of the first chain of reverse transcription reaction or PCR until it is concentrated to 4mg/ml.

2. After homogenization and before adding chloroform, the sample can be stored at -60 ℃ or -70 ℃ for at least one month. RNA precipitation can be stored in 75% ethanol at 2-8 ℃ for at least one week, and at -5-20 ℃ for at least one year.

3. Wear gloves and eye protection when extracting RNA with TRzol LS. Avoid contact with skin and clothing. Complete the operation in the chemical fume hood. Avoid inhalation through the respiratory tract. Unless otherwise specified, maintain the room temperature at 15-30 ℃.

Self provided reagents:

Chloroform, isopropanol, 75% ethanol (prepared with RNase Free water), RNase Free water (add water to a glass bottle without RNase and DEPC to 0.1% (V/V)). Leave overnight and sterilize under high pressure. 0.5% SDS solution (prepared with RNase Free water) (optional).

Operation steps:

1. Sample pretreatment a. Add 0.75ml TRzol LS to every 0.25ml liquid sample (serum, plasma, etc.) of biological liquid, and blow the liquid sample several times with a sample gun to help lyse the cells in the sample. Add at least 0.75ml TRzol LS every 5-10 × 10 6 cells. For samples containing high pollutants such as whole blood samples, they can be diluted twice with sterilized water in a 1:1 ratio before extraction begins. The final volume ratio of TRzol LS to liquid samples is always 3:1. b. Stir the tissue sample evenly with a glass or strong homogenizer, and add 0.75ml of TRzol LS to every 50-100mg of tissue or 0.25ml of tissue suspension. Generally, the volume of 50-100mg tissue should be less than 0.25ml. If the volume of the tissue sample is less than 0.25ml, add sterile water to adjust the volume of the tissue sample to 0.25ml to ensure a volume ratio of 3:1. c. Add 0.3ml-0.4ml of TRzol LS directly to a culture plate with a diameter of 3.5 cm for monolayer growth, dissolve the cells, and use a sample gun to blow to help fully lyse the cells. Determine the required amount of TRzol LS based on the area of the culture plate rather than the number of cells (0.3-0.4ml per 10cm2). There is no need to add water to the lysate, as the residual culture medium attached to the culture plate has already fully diluted TRzol LS. d. Suspended cells are precipitated by centrifugation. Use a pipette to repeatedly blow and lyse cells in TRzol LS reagent. Add 0.75ml of TRzol LS to every 5-10 × 106 animal cells, plant or yeast cells, or every 1 × 107 bacteria. Adjust the sample volume to 0.25ml with sterilized water as in step b. Avoid washing the cells before adding TRzol LS as it increases the possibility of mRNA degradation. Cracking certain yeast and bacteria may require the use of a homogenizer. During the separation stage, incubate the homogenized sample at 15-30 ° C for 5 minutes to facilitate the breakdown of nuclear protein bodies. Add 0.2ml chloroform for every 0.75ml TRzol LS. Cover the sample tube tightly, shake the test tube vigorously by hand for 15 seconds, and incubate it at room temperature for 2-15 minutes. Freeze centrifuge at high speed with a centrifugal force not exceeding 12000 × g for 15 minutes at 2-8 ° C. After centrifugation, the mixture is divided into three layers: the lower chloroform layer, the middle layer, and the upper colorless water sample layer. RNA is present in the aqueous layer without exception. The capacity of the water sample layer is approximately 70% of the added TRzol LS capacity.

2. The precipitation of RNA transfers the water sample layer to a clean test tube. If DNA and protein separation is desired, the organic layer and intermediate layer should also be retained. RNA was precipitated by mixing the water sample layer with isopropanol. Each 0.75ml TRzol LS corresponds to 0.5ml isopropanol. Incubate the mixed samples at 15-30 ° C for 10 minutes and freeze centrifuge at high speed for 10 minutes at 2-8 ° C with a centrifugal force not exceeding 12000 × g. RNA precipitation is usually not visible before centrifugation, but after centrifugation, it forms a gelatinous sheet-like precipitate that adheres to the wall and bottom of the test tube.

3. Rinse RNA to remove the upper suspension. Wash the RNA precipitate once with 75% ethanol (prepared with RNase Free water), and add at least 1ml of 75% ethanol for every 0.75ml of TRzol LS. Vortex oscillation mixed samples are subjected to high-speed freezing centrifugation at 2-8 ° C with a centrifugal force not exceeding 7500 × g for 5 minutes.

4. Dissolve RNA and dry RNA precipitation at room temperature, do not centrifuge and dry RNA in a vacuum tube. It is particularly important not to let RNA precipitate and dry, as this would greatly reduce its solubility. The OD260/OD280 ratio of partially dissolved RNA samples is less than 1.6. Use a pipette to transfer RNase Free water or 0.5% SDS solution (prepared with RNase Free water) in several portions to dissolve RNA (if RNA is to be used for enzyme digestion reactions in the future, avoid using SDS). ）RNA can also be redissolved by 100% formamide (ion removed) and stored at -70 ° C.

What are the basic steps and precautions for PCR?

1、 Experimental principle

PCR is a method of selectively amplifying DNA in vitro, similar to the replication of DNA in living organisms. DNA replication in living organisms requires templates, primers, DNA polymerases, DNA helicases, and dNTPs; And in vitro PCR reactions also require similar components, including templates, primers, PCR buffers, Taq enzymes dNTPs。 Primers are artificially designed specific sequences that achieve amplification at specific positions; PCR buffer provides a buffer environment for the reaction; The reaction process is the same as in living organisms, where the DNA double strand opens, the primer binds to the template, and extends to form a new strand. And these processes rely on DNA helicase to break down the chains inside the organism, while they are achieved outside the body by controlling the reaction temperature. The commonly used 94 ℃ denatured template DNA opens double stranded, primer binds to the template at annealing temperature, and finally extends at 72 ℃, and this process is repeated repeatedly to achieve large-scale amplification of specific DNA fragments. It is not until the third cycle that DNA molecules identical to the target DNA segment are produced, and further cycles result in exponential doubling of the target DNA segment.

In the later stage of amplification, due to product accumulation, the reaction that was originally exponentially amplified becomes a flat curve, and the product no longer increases significantly with the number of cycles, which is called the plateau effect. The plateau period will continue to amplify the low concentration non-specific products that were originally generated due to mismatches, reaching a higher level. Therefore, the number of cycles should be appropriately adjusted to end the reaction before the plateau period and reduce non-specific products. The number of cycles required to reach the plateau depends on the copy of the template in the sample.

2、 Main components

1. Templates can take various forms, including genomic DNA, plasmid DNA, virus carrying genomic DNA, PCR products, cDNA, etc., but cannot be RNA. For different types of templates, the main differences lie in the time of pre denaturation and the amount of templates. Generally, a pre denaturation time of 10 minutes is sufficient for large genomic DNA, 2 minutes for plasmid DNA, 2 minutes for virus carrying genomic DNA, and 2 minutes for PCR product pre denaturation.

Please note that cDNA is a single stranded DNA, but it can still be used as a template for PCR. However, in the first cycle, only one primer binds to synthesize another strand. Starting from the second round, both primers bound to specific sites, achieving alignment with conventional PCR. However, RNA, which is also a single stranded RNA, cannot be amplified by PCR because the PCR reaction is carried out by DNA polymerase, which can only specifically recognize DNA strands.

For the amount of template, the mass of DNA in a typical 25ul system is 50100ng. For genomic DNA, due to its complex structure, the extracted concentration is often high. To prevent excessive concentration from affecting PCR, gradient dilution of the extracted DNA is required. Otherwise, excessive concentration may cause non-specific amplification. For plasmids and PCR products, due to their simple structure and generally low extraction concentration, dilution is not necessary.

What are the issues that should be noted in PCR experiments?

No CT value

The test results encountered a situation where there is no Ct value. Check if there are any of the following issues:

1. The number of cycles is insufficient (generally not exceeding 45 cycles, not only does the background value increase, but the quantification is also inaccurate);

2. The PCR program settings are incorrect, and the steps for detecting fluorescence signals are incorrect. The general SG method uses 72 ℃ extension to collect signals, while the TaqMan method usually collects signals at the end of annealing or during extension. Additionally, is fluorescence collection selected;

3. Primer or probe degradation. The degradation of primers and probes can be detected by PAGE electrophoresis;

4. The template size may degrade or the sample size may be insufficient (not exceeding 500ng, according to the kit instructions). For samples with unknown concentrations, the highest concentration of the series dilution sample should be used as the starting point; If template degradation occurs, the introduction of impurities in sample preparation and repeated freeze-thaw cycles should be considered. It is recommended to pack template samples in small quantities for storage to avoid repeated freeze-thaw cycles;

5. Is the primer probe appropriate (especially when the primer crosses introns to ensure amplification of genomic DNA); Upstream and downstream primer Tm values exceeding 4 ℃ can also affect amplification.

CT value too late

In relative quantification, it is generally better to control the Ct value between 1525. If in absolute quantification, the Ct value will increase for low copy number samples, but it should not exceed 40 cycles, otherwise the quantification will be inaccurate.

Therefore, determining whether the Ct value appears too late is an abnormal situation depends on the specific experimental design and purpose.

1. The amplification efficiency is low. The ratio between primers or between primers and probes is inappropriate and needs to be optimized; Primer or probe design is unreasonable and needs to be redesigned;

2. The PCR program is not suitable. Instead, use a three-step method for the reaction, or optimize the annealing/extension temperature. The annealing temperature can be appropriately reduced; Short annealing/extension time (can be extended by 10 seconds under recommended time conditions);

3. The concentration of MgCl2 is not appropriate, increase the concentration of magnesium ions, etc. Degradation of various reaction components in PCR or insufficient sample addition;

4. The PCR product is too long. PCR product design exceeding 500bp;

5. There are inhibitors present in the template. Perform PCR detection using high-purity templates or dilute the templates.

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