RNAfixer liquid nitrogen free RNA sample storage solution

Product attributes:

Storage conditions:

Store at room temperature (18-25 ℃) for more than 1 year. If precipitation or precipitation is found during use, it can be added to a 37 ℃ water bathAfter hot re dissolution, it will not affect the quality of the product.
Product Introduction
RNAfixer is a liquid, non-toxic tissue preservation liquid that can quickly penetrate into the cytoplasm of fresh tissue cellsIn situ stabilization and protection of intracellular RNA in a non frozen state. Immediately immersing the tissue slices into RNAfixer for preservation does not affect the quality and quantity of RNA extracted in the future. RNAfixer eliminates the need for immediate processing of RNA samplesOr it is inconvenient to store it in liquid nitrogen. After immersion in RNAfixer, RNA in fresh tissue cells can remain intactStore for one day at 37 ℃, one week at 25 ℃, one month at 4 ℃, and long-term storage at -20 ℃ or -80 ℃.
RNA virus samples (such as HCV and HIV) can be stored at 37 ℃ for one month.Suitable for animal tissues (heart, liver, kidney, muscle, testis, brain, spleen, etc.), cultured cells, RNA viruses, fruits, etcFlies, bacteria, white blood cells, some plant tissues, etc.
Product Features:
1. Easy to operate: Organize the tissue into an appropriate size and immerse it in an RNAfixer to prevent its RNA from being degraded.
2. No need for liquid nitrogen: This allows for the preservation of samples without the need for liquid nitrogen, dry ice, or -80 ℃ refrigerators, especially suitable for clinical and field samplesRapid and large-scale collection.
3. Convenient transportation: Processed samples can be stored at 25 ℃ for one week, making sample shipping and transportation easier and cheaper, which is advantageousAcademic cooperation and exchange.
4. Multiple freeze-thaw cycles: Samples treated with RNAfixer can undergo multiple freeze-thaw cycles, during which various treatments can be applied to the samplesWithout affecting the quality of the final extracted RNA.
5. Strong comparability: RNAfixers can reduce errors in large-scale sample processing, increase comparability between experiments, and are particularly useful for analyzing large-scale gene expression profiles.
Instructions for Use:
RNAfixer is only used for fresh tissue, and tissue freezing is prohibited before soaking in RNAfixer. Just need to quickly update the newFresh tissue can be grown, wide, or high, with a thickness of less than 0.5 centimeters on either side, and soaked in RNAfixer (as long as one side is thick)The degree should not exceed 0.5 centimeters, and RNAfixer can quickly penetrate. The size of the other two sides is not important. Fresh groupSoak the fabric in 5 times the volume of RNAfixer and store it at the appropriate temperature as instructed.
1. Animal tissue RNAfixer does not damage or dissolve tissue structure, therefore soaking in RNAfixer achieves penetrationBalanced tissue can be removed from the RNAfixer, then cut into smaller pieces and placed back into the RNAfixerContinue using it next time. Small organs such as mouse liver, kidney, and spleen do not require cutting and can be stored intact in RNAfixers.
Recommended RNAstore usage for different organizational samples:

2. Many plant tissues can be directly soaked in RNAfixers, and some plants have natural permeation barriers such as waxThe protective layer needs to be destroyed first to facilitate the penetration of RNAfixer.
3. After blowing down the cultured cells, collect them by centrifugation, discard the supernatant, and wash them once with PBS buffer in an ice bathRemove residual culture medium. Suspend the cells in a small amount of PBS buffer. Add 5 to 10 times the volume of RNAfixer, mixAll of them.
4. For the preservation of white blood cells in whole blood, white blood cells need to be separated from red blood cells and serum, and grouped accordinglyAfter being treated in the same way as cultured cells, white blood cells can be effectively preserved in RNAfixers. Do not mix whole blood or plasmaOr RNA in serum is stored in RNAfixers because of their high protein content, and after mixing with RNAfixersEasy to form insoluble precipitates.
5. Bacteria
Bacteria cannot grow in RNAfixers, but RNAfixers do not damage bacteria. E. coli can be stored at 4 ℃ for one dayComplete RNA can still be extracted within a month.
Storage of samples in RNAfixer:
1. Store at -80 ℃For long-term storage of samples. Place the sample in the RNA fixer overnight at 4 ℃, then remove the sample as much as possibleRemove the RNAfixer liquid thoroughly and place it at -80 ℃. For tissue cultured cells, there is no need to remove themRNAfixer， Freezing directly at -80 ℃ does not lyse cells. The sample can be melted at room temperature during use, andIt can also be frozen again without affecting the integrity and yield of RNA.
2. Store at -20 ℃Place the sample in the RNAfixer overnight at 4 ℃, and then transfer it to -20 ℃. The sample will not be frozen at -20 ℃Frozen, but may form some crystals, which will not affect future RNA extraction. The sample can be used in the roomWarm melting, and can also be refrozen without affecting the integrity and yield of RNA.
3. Store at 4 ℃The sample can be stored at 4 ℃ for one month.
4. Store at 25 ℃The RNA stored in the sample at 25 ℃ remained intact for one week, while the RNA in the sample stored for two weeks showed slight degradation,It can barely be used for northern analysis, but the quality is sufficient for nucleus protection assay or RT-PCR.
5. Store at 37 ℃The RNA stored at 37 ℃ remained intact within 24 hours, but partially degraded after 3 days.RNAfixer preserves RNA extraction from samples:Remove the sample from the RNAfixer, which can be directly poured into the water tank and rinsed with tap water without the need forSpecial treatment.
1. Organization
Use clean tweezers to remove the sample from the RNAfixer, then use absorbent paper to slightly remove any remaining RNAfixer,RNA can be extracted using the standard procedure of liquid nitrogen grinding and homogenization treatment, just like fresh tissue.
2. Cells
There are two options for cells stored in RNAfixers. One is to remove the RNAfixer and extract RNA, and the other isOne is to extract directly from a mixture of cells and RNAfixers.
1) Extract RNA after removing RNAfixerCells stored in RNAfixers become less fragile and can withstand higher centrifugation speeds without being cleavedSolution. We have experience in successfully collecting cells by centrifugation at 5000g. Due to the varying strengths of each type of cell, we canFirst, conduct a preliminary experiment with unimportant cells to ensure that centrifugation at the appropriate speed will not damage the cells. Another optionIt is recommended to dilute the mixture of RNAfixer and cells with an equal volume of PBS before centrifugation to reduce the density of the solution,Enable the cell solution to precipitate.
2) Extract RNA directly without removing RNAfixer
You can also directly add 10 times the volume of one-step extraction reagents (such as TRIpure, TRI reagent) to cells andMix RNAfixer and follow the normal steps.

PCRBasic steps and precautions?

1、 Experimental principle

PCRIt is an in vitro artificial selective amplificationDNAA method similar to in vivoDNACopy it. in vivoDNACopying requires templates, primersDNAPolymeraseDNAHelicasedNTPsAnd outside the bodyPCRThe reaction also requires similar components, including templates, primersPCR BufferTheTaqEnzymesdNTPsPrimers are artificially designed specific sequences that achieve amplification at specific positions;PCR BufferProvide a buffering environment for reactions; The reaction process is the same as in living organisms,DNADouble strand opening, primer binding to template, extending to form a new strand. And these processes rely onDNAEnzymatic hydrolysis of chains is achieved in vitro by controlling the reaction temperature. As commonly used94℃Transgender TemplateDNAOpen the double strand, bind the primer to the template at annealing temperature, and finally72℃Complete the extension and repeat this process repeatedly to achieve specific fragmentsDNAA large amount of amplification. It wasn't until the third cycle that the target was generatedDNASame sectionDNAMolecules further cyclically generate targetsDNADouble the index of the segment.

In the later stage of amplification,Due to product accumulation,Transform the originally exponentially amplified reaction into a flat curve,The product no longer increases significantly with the number of cycles,This is called the platform effect. plateau period(Plateau)It 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. Arriving at the platform period(Plateau)The required number of cycles depends on the copy of the template in the sample.

2、 Main components

1.Templates can take various forms, mainly including genomesDNAPlasmidsDNAGenome carrying virusesDNAThePCRProduct,cDNAWait, but not forRNAFor different types of templates, the main differences lie in the time of pre denaturation and the amount of templates. Generally for large genomesDNAPre denaturation time10minEnough, plasmidDNA2minGenome carrying virusesDNAPre denaturation2minThePCRProduct pre denaturation2minJust enough.

AttentioncDNAFor single chainDNABut it can still be donePCRThe template only binds to one primer in the first cycle to synthesize another chain. Starting from the second round, both primers bind to specific sites, thus achieving compatibility with conventional primersPCRThe integration. And as a single chainRNABut it cannot be carried outPCRAmplification, the reason lies in the implementationPCRThe reaction isDNAPolymerase, can only be specifically recognizedDNAChain.

2.For the quantity of templates, generally25ulsystemDNAThe quality is50100ngRegarding the genomeDNADue to its complex structure, the extracted concentration is often high. To prevent excessive concentration from affectingPCRCausing impact, therefore it is necessary to extractDNAPerform gradient dilution. Otherwise, excessive concentration may cause non-specific amplification. For plasmids andPCRDue to its simple structure and generally low extraction concentration, the product does not require dilution.

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PCRWhat are the issues that should be noted in experiments?

nonectvalue

Did you encounter any test resultsCtValue situation,Check if there are any of the following issues:

1Insufficient number of cycles(Generally not exceeding45cycle,Not only does the background value increase,Quantification is also inaccurate);

2ThePCRProgram setting error,The steps for detecting fluorescence signals are incorrect. generalSGLaw adoption72℃Collection during extension,TaqManThe rule usually collects signals at the end of annealing or during extension,Also, is fluorescence collection selected;

3Primer or probe degradation. can pass throughPAGEElectrophoretic detection of whether primers and probes degrade;

4The template size may degrade or the sample size may be insufficient(not exceed500ng,Just follow the instructions of the reagent kit),For samples with unknown concentrations, the highest concentration of the series of diluted samples should be used as the starting point;If template degradation occurs,Consideration should be given to the introduction of impurities and repeated freeze-thaw cycles in sample preparation,Suggest to pack template samples in small quantities for storage,Avoid repeated freeze-thaw cycles;

5Is the primer probe suitable(Especially when primers cross introns,To ensure the amplification of the genomeDNA;Upstream and downstream primersTmValue exceeds4℃The above will also affect amplification).

ctValue too late

In relative quantification, CtThe value is generally controlled within1525It's better between them,If in absolute quantification,For low copy number samples,CtThe value will increase,But generally it should not exceed40cycle,Otherwise, the quantification will be inaccurate.

Therefore,judgmentCtIs it considered abnormal if the value appears too late,It needs to be based on specific experimental design and objectives.

1The amplification efficiency is low. Inappropriate ratio between primers or between primers and probes,Optimization is needed;Unreasonable primer or probe design,Need to redesign;

2ThePCRInappropriate program,Switching to a three-step reaction method,Or optimize annealing/Extended temperature,The annealing temperature can be appropriately reduced;annealing/Short extension time(Can be extended under recommended time conditions10s);

3TheMgCl2Inappropriate concentration,Increase the concentration of magnesium ions, etc.PCRDegradation of various reaction components or insufficient sample size;

4ThePCRThe product is too long.PCRProduct design exceeds500bp;

5There are inhibitors present in the template. Using high-purity templatesPCRDetect or dilute the template.