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Questions about in vivo animal experiments

Q:Can I use siRNA without being chemically modified in animal experiments?

A:Unmodified siRNA can be used for animal experiments with special transfection reagents or medication methods. However, animal experiments have high requirements on the stability and half-life period of siRNA reagents in vivo, hence specially chemically modified or in vivo-grade reagents are recommended.

Q:Which type of chemical modification can be used for siRNA in vivo experiments?

A:RiboBio provides a variety of chemical modification methods like 2’OMe, FU/FC, PS, we also provide specific siRNA for animal experiments.

Q:Modified siRNA experiment has been completed at the cellular level, can the remaining siRNA be used for animal experiments?

A:It is generally not recommended to use the remaining siRNA reagents for animal experiments. As the in vivo animal experiment conditions are more complicated, it requires special treatment to synthesize siRNA, therefore, more reliable siRNA for animal experiments are recommended.

Q:What is the difference between the three kinds of siRNA (Standard, in vivo; PAGE, in vivo; HPLC, in vivo) used in animal experiments?

A:All the three siRNA are treated or purified according to animal experiment standards, and can be used for in vivo animal experiments. The difference lies with purity: the full-length RNA content of Standard siRNA is no less than 80%, and the full-length RNA content of PAGE and HPLC siRNA is no less than 95%. The higher the purity, the better the specificity and targeting, the more reliable the effect, but also the higher the cost. Therefore, if budget permits and it has higher requirement, siRNA with higher purity are recommended.

Q:Can we reference any published articles using RiboBio's siRNA for animal experiment?

A:Yes, please contact our business staff.

Q:How do you medicate different organs? What is the recommended amount and frequency for medication?

A:The medication method is roughly classified into systemic administration (intravenous administration, intraperitoneal administration, etc.) and topical administration (subcutaneous injection, vitreous administration, intrathecal administration, etc.) according to different organs. The dosage is also different depending mainly on the weight of the subject. Intravenous injection (systemic administration) is generally involved in liver and kidney and intratumoral injection (local administration) in subcutaneous tumors; 5~20nmol/20g is required for systemic administration and 1~5nmol/20g for local administration. The frequency of administration is 2~3 times per week.

Q:How much siRNAs is needed for mice in vivo studies?

A:It is necessary to determine the amount and frequency of administration based on the animal model and experimental scheme, and then calculate the total amount required for the experiment. Generally. 5~20nmol/20g is required for systemic administration and 1~5nmol/20g for local administration; the frequency of administration is 2~3 times per week.

Q:How to medicate miRNA agomir/antagomir for different organs? What is the recommended amount and frequency for medication?

A:The medication method is roughly classified into systemic administration (intravenous administration, intraperitoneal administration, etc.) and topical administration (subcutaneous injection, vitreous administration, intrathecal administration, etc.) according to different organs. For example, the liver and kidney are usually intravenously administered (systemic administration); while subcutaneous tumors are intratumorally administered (topical administration). Systemic administration: agomir injection at 5~20nmol/20g, antagomir injection at 50~200nmol/20g each time; topical administration: agomir injection at 1~5nmol, antagomir injection at 5-20nmol each time. The frequency of administration is 2 to 3 times per week.

Questions about cell transfection experiments

Q:How to choose transfection methods and transfection reagents?

A:The selection of transfection methods and transfection reagents depends on specific cells. riboFECT CP transfection reagent is recommended for easily transfected cells, riboFECT mRNA transfection reagent for mRNA/long-chain RNA transfected cells, primary cells or difficult-to-transfect cells, and riboFECT Protein transfection reagent for efficient protein transfection. RiboBio provides a full range of transfection reagents from siRNA/miRNA, mRNA/long-chain RNA to proteins, please choose the appropriate transfection reagent according to the Table below.

siRNA/miRNA/Plasmid transfection reagent, high gene suppression efficiency mRNA/long-chain RNA transfection reagent for primary cells Protein transfection reagent for the most efficient transfection
riboFECT™ CP transfection reagent riboFECT™ mRNA transfection reagent riboFECT™ Protein transfection reagent
Unit 0.5ml, 1ml 0.5ml, 2.5ml 0.5ml, 2.5ml
Sample Type siRNA, miRNA mRNA, longRNA Protein
Transfection Efficiency Extremely Good Extremely Good Highest, can reach 98%
Cell viability Extremely Good Extremely Good Good
Cytotoxicity Extremely Good Extremely Low Almost no cytotoxicity
Cell Lines HeLa,A549,HEK293T,HCT-116,NIH/3T3,HepG2,HNE1,L6,5637,H-bc,HSC,C2C12,Sertoli,CNE-2,5-8F,HAEC,DRG,CF,THP-1,MEL,PBMC,NSC,MSC,U2OS human fibroblasts,CHO-K1,B16-F10,HEK293 HEK293,B16-F10,COS-7,COLO 205,dendritic cells (DC),H9c2,human fibroblasts (primary cells),HUVEC (primary cells),Jurkat,MC3T3-E1,MEF (primary cells),microglia,mouse podocytes (primary cells),NIH/3T3,SK-OV-3,U-373 MG
Q:How to improve the transfection efficiency for cells that are difficult to transfect? How to determine the transfection efficiency?

A:For adherent cells, transfection reagent is recoomended for transfection; for difficult-to-transfect cells, electroporation is recommended, but the riboFECT mRNA transfection reagent is also recommeded to minimize the damage of cells. Fluorescence-labeled siRNA is often used to detect transfection efficiency with fluorescence microscopy, confocal microscopy or flow cytometry, please refer to the product manual for details.

Q:Is the transfection efficiency of cells related to siRNA sequence?

A:The efficiency of transfection depends on the cell itself and the transfection method, it has no direct relation with the siRNA sequence.

Q:What is the most appropriate cell density for siRNA transfection?

A:It depends on the transfection method or transfection reagent. If siRNA is transfected with riboFECTTM CP Reagent, the optimal cell density is 50%-80%.

Q:Can serum be contained in the medium required for transfection?

A:It varies for different transfection reagent. For riboFECT™ CP Reagent, serum should not be included in the preparation of siRNA and riboFECTTM CP mixture; cell culture medium may contain serum but not antibiotics.

Q:What is the difference between siRNA storage solution concentration and working concentration?

A:The storage concentration of siRNA is the optimal concentration for preservation, and it’s recommended at 20μM; the working concentration of siRNA is generally in the range of 10-100 nM to achieve the best silencing effect. We recommend the transfection concentration at 50nM.

Q:How to divide groups for transfection? What's the purpose of grouping?

A:

A组(必需)针对目的基因siRNA(重复三个孔)knockdown目的基因的表达
B组(必需)阴性对照siRNA(重复三个孔)用非特异的siRNA序列证明RNAi作用的序列特异性
C组(必需)转染试剂对照(重复三个孔)排除转染试剂对细胞的毒性或对目的基因表达的影响
D组(必需)空白对照(重复三个孔)用于检测实验过程中细胞的生长状态
E组(可选)阳性对照siRNA(重复三个孔)用于排除实验体系和实验操作的问题
F组(可选)荧光对照siRNA(重复三个孔)用于转染效率。当不明确细胞转染效率时,这一组为必需。

Q:How to detect transfection efficiency with fluorescent-controlled siRNA? Is it necessary for every experiment?

A:RiboBio’s transfection-controlled siRNA are labelled with Cy3 or Cy5 fluorescent, which can be detected by fluorescence microscopy, confocal microscopy, flow cytometry and other fluorescence detection instruments. In addition, positive control experiments should also be conducted for further detection. The transfection efficiency is mainly related to the cells themselves, and it should be similar under equivalent conditions, so there’s no need to do it every time. However, a fluorescent control group for each experiment is able to exclude potential problems.

Q:What is the cause of cell death after transfection? How to optimize transfection conditions?

A: If the transfection reagent is toxic to cells, it’s recommended to reduce the amount of transfection reagent or switch to other transfection reagents or transfection methods; if the cells are not in good condition, it is recommended to re-test and resuscitate the cells if necessary.

Q:What if the cell density is too low at the due time of transfection? Should we wait for another day for the cells to be transfected?

A:Cell growth requires a certain density, and the transfection reagent is toxic to cells. If the cell density is too low during transfection, the cells may grow abnormally or even die. Good cell status and cell viability are required before transfection, so it is generally not recommended to extend cell growth for transfection.

Questions about positive/negative control experiments

Q:Why is a positive control important in RNA interference experiments?

A:Positive control is an inspection of the experiment system. In other words, a siRNA positive control with expected results can ensure the reliability of transfection, RNA extracts and detection methods.

Q:What is the role of positive control and negative control in RNAi experiments?

A:A positive control refers to an siRNA that has been validated against a housekeeping gene or a reporter gene, and is used to monitor the feasibility of experimental systems and experimental methods. RiboBio provides siRNA against GAPDH, ACTB, GFP/EGFP for positive control. Negative controls are often non-specific siRNAs mainly used to illustrate the specificity of siRNA function, universal or random sequences are available.

Q:Should the results of each group of negative controls be the same? What if the deviation is larger than expected?

A:Under normal circumstances, the results of each group of negative controls should be similar, and accuracy should be a primary criterion in case of larger-than-expected deviation. In addition, genes associated with the stress of cells and genes involved in cellular immunity may be sensitive to external pressure, thus resulting in inconsistent gene expression changes.

Questions about RNAi experiments

Q:What's the wavelength of siRNA fluorescent dye with the maximum absorption rate and emissivity?

A:FAM has a maximum absorption at 495nm and a maximum emission at 520nm; Cy3 has a maximum absorption at 550nm and a maximum emission at 565nm; Cy5 has a maximum absorption at 643nm and a maximum emission at 667nm.

Q:How to test the siRNA effects?

A:The effects of siRNA can be tested from three aspects. 1) to detect mRNA levels with Realtime RT-PCR; 2) to detect protein levels with Western blot, ELISA, etc.; 3) to detect cell phenotype with high-intensity microscope according to the function of the gene of interest

Q:How long does siRNA work in cells? When is the best time to detect?

A:siRNA-mediated RNAi is a transient phenomenon and cannot be stably subcultured. Since the effective period cannot be maintained for long, it’s recommended to complete the detection within 3-4 days post transfection. The optimal detection time varies depending on the cell and the target gene, and it’s mostly conducted within 24-48 hours after transfection.

Q:Why should we emphasize the detection of mRNA levels? Can proteins and functions be directly detected?

A:siRNA acts directly on mRNA, making mRNA level the most obvious indicator. As the direct degradation of mRNA is believed to lead to a corresponding decrease of protein content, the detection of protein levels should also be used as a measure of effectiveness. In fact, the mRNA decline does not necessarily correspond to protein decline. Possible reasons can be: 1) Detection time. If the decrease in mRNA is not reflected in the amount of protein or has not reached the detection level, it is generally recommended to delay the detection; 2) the expression of protein in cells. The translation process of mRNA is very complicated, and the expression of genes in cells always maintains an equilibrium. After a certain amount of protein is expressed to a certain extent, it is enough to maintain its cellular function, and may temporarily “close” the function of expression, thus part of the transcribed mRNA may not be involved in the process of protein translation, so the decline of mRNA levels is not entirely positively related to the decline of protein levels; 3) more complex mechanisms.

Q:How high should the interference efficiency be to make a good target?

A:There is no clear standard, as the efficiency of interference varies with different cell types and genes. The transfection efficiency of different cells is varied, and the expression levels of different genes are also quite different, mainly depending on the interference effect.

Q:What if the silencing effect didn't turn out as expected?

A:Low transfection efficiency and poor siRNA sequence design are the two most common reasons behind bad silencing effects. If you are using siRNA for the first time or using a new cell line and found that the silencing effect is not good, we suggest that you test the transfection efficiency and optimize the transfection condition. If the problem persists, it’s recommended that you switch to other transfection reagents or techniques to improve transfection efficiency. Otherwise, it may be attributed to the poor siRNA sequence design.

Q:Why does siRNA upregulate the target gene expression? How to deal with it?

A:Possible reasons include: 1. Excessive transfection concentration or inappropriate transfection conditions may lead to abnormal gene expression, it’s recommended to optimize transfection conditions; 2. Some type of cells may be sensitive to external incentives, it’s recommended to switch to other cells; 3. Inaccurate test results, it’s recommended to optimize the test conditions (RNA quality, primer design, reaction conditions, etc.)

Q:What if a negative control experiment presents the same results as a specific RNAi experiment? What does this mean?

A: Possible causes include: 1. Results of non-specific effects: Abnormal gene expression may be caused by excessive transfection concentration or inappropriate transfection conditions, it’s recommended to optimize the transfection condition; 2. Negative control siRNA sequences have presented unexpected off-target effects, and interfered with the gene of interest, it’s recommended to switch to other negative control siRNAs.

Q: If only 50% silencing efficiency was obtained in the transfection of 100nM siRNA, is it possible to increase the siRNA concentration to 200nM-400 nM for better silencing efficiency?

A:To improve interference efficiency, it’s recommended to optimize the transfection concentration to the range of 10~150nM. Excessively high concentration of siRNA may increase the possibility of non-specific action and may be toxic to cells.

Q:Why is the same siRNA effective in cell A but not effective in cell B?

A:Different cells have different transfection efficiencies and gene expression levels, both are related to the efficiency of siRNA.

Q:How to design siRNA?

A:RibiBio provides  siRNA design if gene name or gene ID provided by the customers.

Q:Is the terminal overhang of siRNA necessary? How to choose?

A:The traditional siRNA structure is a 19 mer + 2′ overhanging structure, and some siRNAs are currently not blunt-end. dTdT/UU is the most common choice for drape, and dNdN (N stands for the base corresponding to the gene sequence) is also used as a drape. The choice of overhang does not significantly affect the efficiency of siRNA.

Q:Is it possible to design a siRNA that targets a protein if you know the protein sequence of a certain gene product?

A:siRNA acts on the RNA level, instead of the protein level. The design of a siRNA requires an accurate target mRNA sequence. Due to the degeneracy and shift of genetic code (codon), it’s impossible to accurately predict the nucleotide sequence by the protein sequence. The post-transcribed RNA precursor removes the intron sequence to form mature mRNA by splicing. Since the function of siRNA is to hydrolyze the mRNA sequence, the RNA sequence designed according to the genomic sequence may fall into the intron region, resulting in the ineffective design of siRNA, so the siRNA should be designed based on the mRNA sequence rather than the genomic sequence.

Q:Why is there an AA structure at the 5' end of the published siRNA sequence? Is this structure necessary?

A:“AA” used to represent the beginning of a siRNA target sequence, and is often used as part of the sequence composition but not part of the synthetic siRNA sequence. The siRNA target sequence is often written in the form of “AA-(N19)”, where “AA” indicates that the 3′ end is suspended as dTdT or UU. The N19 target sequence and the two overhangs combine to form a 21-mer duplex. AA structure is not necessary for siRNA design.

Q:Is the siRNA designed for human genes effective for other species as well?

A:Even though the genes of different species are homologous, the expression sequences of these homologous genes are more or less varied; and as siRNA requires sequence specificity, ie, complete complementary pairing, most siRNAs cannot achieve the gene silencing effect for multi species. Even for the same fragment of homologous genes of different species, the gene silencing effects in different species cannot be guaranteed due to the difference in gene expression.

Q:Can the chemically synthesized siRNA target sequences be used to construct shRNA eukaryotic expression plasmid vectors?

A:Yes, but different shRNA expression vectors have different requirements for the sequence, so the feasibility depends on the specific requirements of the expression vector.

Q:Will an effective sequence determined by chemical synthesis stay so after being constructed in a shRNA expression vector?

A: It should be as effective as before, but because of the complexity of vector expression, it depends on whether the vector can be strongly expressed in cells, or whether it can be processed correctly, etc. Therefore, the possibility of shRNA turn ineffective cannot be excluded.

Q:Can the effective sequence of shRNA expression vectors remain equally effective with chemical synthesis?

A:The method of shRNA expression vector is prone to “false positive”, and it does not rule out the possibility that siRNA turn ineffective.

Q:Can the effective sequence of shRNA expression vectors remain equally effective with chemical synthesis?

A:The method of shRNA expression vector is prone to “false positive”, and it does not rule out the possibility that siRNA turn ineffective.

Q: Is low-temperature transport required for siRNA?

A:RiboBio’s siRNA is supplied in the form of lyophilized powder that can be shipped at room temperature, and should be stored at -20~-80°C after receipt.

Q:The delivery took nearly a week, will siRNA degrade after being exposed at room temperature for such a long time?

A:siRNA in the form of lyophilized powder can be stably stored at room temperature for 1-2 weeks. 

Q:How to dissolve and store siRNA?

A:Please centrifuge siRNA before opening the lid, add sterilized RNase-free H2O or sterilized ddH2O to prepare 20μM stock solution, then separately store the solution at -20°C-80°C.

Q:To prepare a storage solution with a concentration of 20uM, how do I calculate the amount of suspended siRNA buffer?

A:The siRNA provided by RiboBio is lyophilized powder measured in nmol. The sample concentration (umol/L) equals the amount of siRNA (nmol) divided by resuspension volume (uL). For example, to dissolve 5nmol of siRNA into a 20uM sample, 250uL of buffer should be applied. Specific dissolution volume can be seen in the following table:  Table 1. Preparation reference for 20μM stock solution

siRNA(nmol)0.250.512520
溶解体积(μl)12.525501002501000

Q:Will the repeated use affect the quality of siRNA, which is dissolved and stored at -20°C?

A:It’s suggested that the solution be separately stored after dissolving, and it’s not recommended to freeze and thaw the siRNA in solution for more than 5 times.

Q:Does siRNA need to be annealed before use?

A:RiboBio’s siRNA has been annealed and can be used directly.

Q:How to determine the transfection rate of miRNA mimic and inhibitor?

A:The most direct approach is to add a transfection control and observe the transfection rate.

Q:During transfection, is the concentration of miRNA mimic and inhibitor the same?

A:Due to the competitive inhibitory mechanism of miRNA inhibitor, it often requires a large amount to achieve the expected inhibitory effect, which is several times the amount of mimic. The recommended transfection concentration is 50 nM for mimics and 100 nM for inhibitor.

Q:How to co-transfect multiple miRNAs?

A:The co-transfection can be carried out with a conventional method according to the transfection concentration of each miRNA.

Q:Why is the result of miRNA mimic experiments never repeated?

A:It may be related to the spatial and temporal specificity of miRNA expression and function.

Q:What is the difference between miRNA inhibitor and its miRNA antagomir? How to choose?

A:Both miRNA inhibitor and antagomir are used to study the function of inhibited miRNA, their difference lies mainly in the modification method. Both are optional for cell experiments, but antagomir is relatively popular for animal experiments.

Questions about qPCR experiments

Q:How to design the repetition of miRNA qPCR experiments?

A:The repetition is divided into biological repetition and technical repetition. The biological repetition is the independent experiment from the time of re-extracting RNA after processing the sample; technical repetition can be conducted in either cDNA synthesis or PCR reaction, mainly depending on whether there is any problem in the experimental operation process. The repetition is usually conducted three times in PCR.

Q:How large is the product of 5S qRT-PCR?

A:91 bp, the 5S of human, mouse and rat are homologous.

Q:How large is the product of U6 qRT-PCR?

A:94 bp, the U6 of human, mouse and rat are homologous.

Q:How large is the product of Bulge-LoopTM miRNA qRT-PCR?

A:About 80 bp, the length of qRT-PCR products of different miRNAs is varied, depending on the actual primer length.

Q:Why is NTC amplified?

A:Possible causes can be: 1) Reagent contamination; 2) Primer dimer Solution: 1) Switch to other reagents; 2) Reduce primer concentration; 3) Adjust program settings like increasing annealing temperature or reading temperature.

Q:Why is there a heterogeneous peak in non-specific amplification?

A:Possible causes can be: 1) RNA is contaminated by genomic DNA; 2) the primer is mixed up with other primers; and 3) the existance of primer dimer Solution: 1) To extract higher quality RNA and treat sample with DNaseI; 2) To prepare reaction system on ice and reduce initial non-specific amplification; 3) To adjust the reaction system, such as reducing the concentration of primers and the amount of starting template; 4) To adjust the program settings like increasing the annealing temperature and the reader temperature; and 5) To replace the contaminated RNA templates, primers, enzymes, etc.

Q:What if the product of qPCR is less than expected?

A:Possible causes can be 1) Low RNA template quality or cDNA template quality; 2) Low or zero expression of target miRNA gene making it unable for detection; 3) PCR inhibitor in reagents; 4) improper function of PCR instrument; 5) Problems in PCR procedure setting, such as insufficient number of cycles, no process of reading, no process of making melting curve, etc.; 6) Sybr, forward and reverse primers, templates or other reagents are missing in the PCR process; 7) Invalid enzymes, sybr and other reagents used in the PCR process; 8) Problems with quantitative PCR primers like low concentration, poor primer design, incorrect primer synthesis sequence. Solution: 1)To add a positive RNA control test; 2) To increase the amount of RNA and/or cDNA template; 3) To ensure the normal operation of quantitative PCR instrument; 4) To adjust the program settings, such as increasing the number of cycles, the normal plate reading process and dissolution curve preparation process; 5) To set the preheating process according to instructions, as the enzyme used in PCR reaction is generally hot start polymerase; 6) To ensure that no reagents are added or missing during the preparation of the PCR reaction system, and that the reagents are effective; 7) To ensure the content of the reaction product of the first strand is no more than 1/10 in the process of PCR amplification.

Q:How to deal with low cDNA yield?

A:Possible causes can be: 1) Low RNA template quality; 2) Excessively high estimate of mRNA concentration or inaccurately tested concentration; 3) Reverse transcriptase inhibitor or insufficient reverse transcriptase in the reaction system; 4) Reagents missed in the reaction system; 5) Incorrect temperature control process, high temperature may lead to the failure of reverse transcriptase, low temperature makes it unable to reach the best reaction efficiency of reverse transcriptase; 6) Excessive reaction volume resulting in reagent dilution Solution: 1) Re-extract high quality RNA template and test its concentration; 2) Add various reagents to establish a complete reaction system; 3) Ensure correct temperature control process and 4) Reverse transcription reaction system should not exceed 50uL.

Q:Why did the amplification curve descend after rising normally? Why is the dissolution curve irregular?

A:An enzyme inhibitor in the reaction system or other reasons may lead to the inactivation of PCR enzyme or dye degradation. It is recommended to re-examine the RNA template and replace the PCR reagent for re-experiment.

Q:After the transfection of mimics or inhibitor, how to use qPCR primers to detect the changes in the amount of miRNA after transfection? What should we pay attention to?

A:For cells with high transfection efficiency (hela, A549, etc.), 50nM transfected mimics is about 10,000 times higher than no-target, and the plasmid expressing of transfected miRNA is hundreds higher than the blank control plasmid. The inhibitor bound to miRNA is able to theoretically prevent its binding to the target mRNA, and the inhibitor does not cause the degradation of miRNA, therefore, qPCR can detect the increase of miRNA expression after mimics transfection, but not the decrease of miRNA expression after inhibitor transfection. However, if some inhibitors do cause miRNA degradation through unknown ways, a decrease in miRNA expression can also be detected. There is no need for speacial in the RNA extraction and RT-PCR process, except to ensure the transfection efficiency during transfection.

Q:Should the reverse transcription reaction of the internal reference and miRNA be carried out in the same tube or in a separate tube? Why?

A:Internal reference is mainly set to verify the difference in the amount of the sample, so as to ensure that the genes are compared under the same condition. There is no substantial difference between the two methods if the amount of the sample involved is the same. Generally, a reverse transcription is able to detect all the mRNA genes, so reverse transcription is suggested to be performed in the same tube. Due to the limited length and small scale of miRNA, each miRNA needs to be reverse transcribed with specific reverse transcription primers. If the primers cannot be mixed together, the reverse transcription of the internal reference should be carried out separately.

Q:Can I mix use different miRNA reverse transcription primers?

A: The mixed use of various primers may lead to the production of primer dimers and non-specific amplification, therefore, pre-experiments should be carried out to compare the effects of the mix on miRNA quantitative detection. It can only be mixed if no effects were indicated. It is recommended that each miRNA be separated from reverse transcription and quantitative PCR.

Q:Can I mix use different miRNA reverse transcription primers?

A:The mixed use of various primers may lead to the production of primer dimers and non-specific amplification, therefore, pre-experiments should be carried out to compare the effects of the mix on miRNA quantitative detection. It can only be mixed if no effects were indicated. It is recommended that each miRNA be separated from reverse transcription and quantitative PCR.

Q:What's behind the varied peaks for different miRNA dissolution curves and different RNA template dissolution curves amplified by the same miRNA primer?

A: The dissolution curve only appears when the dye is used for detection, and detections conducted with Taqman probes don’t include the drawing process of dissolution curves. The dissolution curve is drawn with the in-line dye binded to all double-stranded DNA (dsDNA) in the system, including target products, non-specific amplification products and primer dimers. As the temperature slowly rises from below the dissolution point to above the dissolution point, the product gradually melts, the in-line dye gets released, and the fluorescence value of the sample monitored by the real-time instrument is continuously descending and it decreases most fast at the dissolution point of the product, thus creating a peak on the dissolution curve. After the complete melting, the fluorescence value will reach a plateau. The length of amplified products, G/C content, pH value, ion concentration, protein residue, etc. all affect the peak value. For the same primer to amplify different templates, a peak deviation within 1°C can be neglected. Otherwise, it is necessary to redo the experiment if problems of primers and templates are found. For different primers with a single peak, its’s resonable to take no account of the peak difference.

Q:Are there special requirements for enzyme and other reagents used in experiments?

A:Primers have no special requirements for enzymes, reagents or instruments, only that the experiments should be carried out with the same instrument, reagent and procedure. Different reagents from different manufacturers, different batches of reagents from the same manufacturer, and different instruments may all slightly affect the amplification curve and the dissolution curve, in spite of no effect on the relative quantification. For reagents from different manufacturers or different instruments, it is recommended to do a preliminary experiment to test the feasibility of reaction system and program. If the result is not satisfactory, appropriate adjustment can be made according to the reagent description and instrument parameter setting principles.

Q:Why is the PCR amplification efficiency lower than expected?

A:Theoretically, the most ideal PCR amplification efficiency is close to 100%, and the amplification efficiency should be at 90%-110% in practice. Amplification efficiency E=10-1/Rate of slope-1, where the rate of slope is the slope of the standard curve and should range from -3.1 to -3.5. Lower-than-expected amplification efficiency may be caused by PCR enzyme inhibitor, invalid PCR dye, improper primer design, unoptimized reaction conditions, etc. When the amplification efficiency is greater than 100%, possible reasons can be the calculation or loading error, the production of non-specific amlification or primer dimer in serial sample dilution.

Q:How to design control groups in PCR?

A:Positive control: The cDNA reverse-transcribed by RNA is generally used as a template to detect housekeeping genes such as β-Actin and GAPDH. The purpose is to find problems related to the configuration of the reaction system, the parameters of the instrument during the reverse transcription and PCR process, and the operation process. Also, plasmid can also be used as a template for quantitative PCR to quantitatively amplify the cloned product, and detect problems involved in the PCR reaction system and the operation process. Negative control: Water can be used as a template for negative control to detect whether the reagents used in the reaction system are contaminated. RNA can be used as a template for negative control to detect DNA contamination during RNA extraction. Internal reference test: The expression of housekeeping genes such as β-Actin and GAPDH can be detected for internal reference.

Q:Is there any special requirement for RT reaction in Bulge-Loop, qRT-PCR Primer?

A:No. RT reaction can be conducted with regular kits by replacing the RT primer with Bulge-LoopTM RT primer. Please refer to RT reagent operating instructions for specific information.

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