Question 1

What are the common methods of oligonucleotide purification?

Accepted Answer

The common purification methods are DSL, OPC, PAGE, HPLC and Dual PAGE & HPLC.

Question 2

Will the primers degrade when transported at room temperature?

Accepted Answer

No, they will not degrade. Dry primers can be stably stored at room temperature for at least two weeks. Since the average transport time is typically 5 days, the primers you receive will not be degraded.

Question 3

How many OD units of primer should be synthesized?

Accepted Answer

The required OD value depends on the experimental purpose.* 2 OD of a 20-base primer is sufficient for approximately 400 standard 50 μL PCR reactions.* For applications like gene splicing or annealing for ligation, 1 OD is often sufficient.

Question 4

What are the basic principles of primer design?

Accepted Answer

Key considerations for effective primer design include:(1) Length: Generally between 15 and 30 bases.(2) GC content: Between 40% and 60%.(3) Base distribution: Bases should be randomly distributed.(4) Complementarity: There should be no complementary sequences within the primer itself (hairpins) or between the two primers (primer dimers).(5) Binding energy (∆G): Primers should have relatively high ∆G values at the 5' end, middle ∆G values, and low ∆G values at the 3' end.(6) Product structure: The single strand of the amplification product should not form a secondary structure.(7) Specificity: The primers must have high specificity for the target.

Question 5

What causes low fluorescent signal in amplification curves?

Accepted Answer

Low signal values are mostly due to high background fluorescence (for example, check the baseline-corrected or ROX-corrected raw curves). In probe-based detection, this is often caused by:        * Poor probe design: Leading to insufficient quenching by the quencher group.* Improper pairing: Incorrect matching of the reporter and quencher groups.* Low labeling efficiency: The probe's fluorescence labeling efficiency is too low.

Question 6

What is the principle of oligonucleotide synthesis and what types of services are offered?

Accepted Answer

oligonucleotide synthesis utilizes solid-phase phosphoramidite chemistry. We synthesize a wide range of oligonucleotides, including:(1) DNA oligos(2) RNA oligos(3) Probes(4) siRNA(5) sgRNA(6) miRNA(7) ASOs(8) Various modified oligos

Question 7

What contaminants are present in the crude product?

Accepted Answer

Crude oligonucleotides contain the desired product, failure sequences (shorter strands), and chemically modified by-products.

Question 8

How to store oligonucleotides?

Accepted Answer

(1) Storage (Freeze-Dried): oligonucleotide primers in a freeze-dried state are highly stable and can be stored at -20°C for at least one year.(2) Storage (Dissolved): Dissolved oligonucleotides can be stored as a 100 μM stock solution, aliquoted into multiple tubes, and kept at -20°C for at least six months (frequent freeze-thaw cycles may reduce shelf life).Before use: Dilute the concentrated stock solution to the working concentration required for your experiments.

Question 9

How to dissolve oligonucleotides?

Accepted Answer

Before opening the vial, centrifuge it at 3000–4000 rpm for 1 minute to ensure the powder is at the bottom and prevent loss.Add an appropriate amount of nuclease-free double-distilled water (pH > 6.0) or TE buffer (pH 7.5–8.0) to achieve a 100 μM stock solution for each OD.

Question 10

Why do resuspended primers perform well initially but degrade after storage?

Accepted Answer

Primer degradation occurs if the dissolving water has a low pH or is contaminated with bacteria or nucleases. Inadequate thawing and mixing when using the aliquot may also lead to inaccurate primer addition.Recommendation:* Aliquot the primers to avoid repeated freeze-thawing.* Use 10 mMTris buffer (pH 7.5) to dissolve the primers.* Note that sometimes the primer itself is not faulty, but the quality of other PCR materials (especially the template) may not be the same as previously used.

Question 11

Why do long chain primers cost more than short chain primers?

Accepted Answer

Synthesizing long-chain primers requires more reagents, especially for primers longer than 90 bases, which need combining. Due to the increase in reagent and synthesis costs, the price is higher.

Question 12

What customization options are available for custom DNA oligonucleotides?

Accepted Answer

The entire process from synthesis, QC to packaging can be highly customized. Customizable options include, but are not limited to:(1) Packaging: In 1.5 mL, 15 mL, or 50 mL tubes.(2) QC analysis: Capillary electrophoresis analysis, HPLC, PAGE, qPCR, full-wavelength scanning, and OD260/OD280 measurement.(3) Documentation: Customized Certificate of Analysis (COA).(4) Other: Provision of a 1 OD sample with each shipmentand specialized consumables.

Question 13

Sometimes the dried primers are yellowish-brown, is this the color of DNA itself?

Accepted Answer

Synthetic primers may appear yellowish-brown, white or transparent. This color is related to the base composition and the synthesis process. Primers with high A and G content and large OD value are usually yellowish-brown. Yellowish-brown primers will not have any effect on the experiment.

Question 14

How to test the purity of primers?

Accepted Answer

A common laboratory method is PAGE. Since primers are single-stranded DNA and can form complex secondary structures, denaturing PAGE is essential.Gel Recommendations:* Primers <12 bases: 20% polyacrylamide gel (7M urea)* Primers 12 - 60 bases: 16% polyacrylamide gel (7M urea)* Primers >60 bases: 12% polyacrylamide gel (7M urea)Notes:(1) Dissolve 0.2-0.5 OD primer with saturated urea solution (or add dry urea powder into primer solution until saturated) and heat denature (95°C, 2 min) before loading. The urea denatures the DNA and increases sample specific gravity for easier loading.(2) Detect the bands with a fluorescent TLC plate under a UV lamp. Good purity is indicated by no impurities below the main band. (Sometimes due to insufficient denaturation, there may be bands above the main band, which are primer secondary structure bands.)

Question 15

How to measure the OD value of primers?

Accepted Answer

The OD value of a synthetic primer is determined by using a UV spectrophotometer at a wavelength of 260 nm and a 1 cm path length quartz cuvette.* The solution must be diluted so that the optical density is between 0.2-0.8.* The OD value of the stock solution must be calculated by multiplying the reading by the dilution ratio.For example, the DNA was dissolved in 1 mL of water to create a stock solution. A 1:10 dilution of this stock (100 μL stock + 900 μL water) yielded an A260 of 0.2 when measured in a 1-cm path length quartz cuvette. This measurement corresponds to an original stock concentration of 2 OD.

Question 16

When PAGE electrophoresis is performed, why are the swim bands of Oligo DNA of exactly the same length not in the same position?

Accepted Answer

The OD value of a synthetic primer is determined by using a UV spectrophotometer at a wavelength of 260 nm and a 1 cm path length quartz cuvette.* The solution must be diluted so that the optical density is between 0.2-0.8.* The OD value of the stock solution must be calculated by multiplying the reading by the dilution ratio.For example, the DNA was dissolved in 1 mL of water to create a stock solution. A 1:10 dilution of this stock (100 μL stock + 900 μL water) yielded an A260 of 0.2 when measured in a 1-cm path length quartz cuvette. This measurement corresponds to an original stock concentration of 2 OD.

Question 17

Why does the EB staining vary in shade for the same OD detected by PAGE?

Accepted Answer

The OD value of a synthetic primer is determined by using a UV spectrophotometer at a wavelength of 260 nm and a 1 cm path length quartz cuvette.* The solution must be diluted so that the optical density is between 0.2-0.8.* The OD value of the stock solution must be calculated by multiplying the reading by the dilution ratio.For example, the DNA was dissolved in 1 mL of water to create a stock solution. A 1:10 dilution of this stock (100 μL stock + 900 μL water) yielded an A260 of 0.2 when measured in a 1-cm path length quartz cuvette. This measurement corresponds to an original stock concentration of 2 OD.

Question 18

What is the longest primer that can be synthesized?

Accepted Answer

As oligonucleotide length increases, synthesis complexity and the risk of incomplete sequences also increase. Unless specifically required, we recommend limiting primer length to 80 bases or less. At this length, the proportion of full-length product in a crude synthesis typically does not exceed 40%, and additional losses may occur during purification, resulting in reduced final yield.With optimized synthesis conditions, primers of up to 145 bases can be produced; however, yields and purity decrease significantly at extended lengths, and such designs should be considered only when shorter alternatives are not feasible.

Question 19

Can I use Agarose gel electrophoresis to analyze the synthetic primers?

Accepted Answer

Agarose gel electrophoresis is unsuitable for analyzing synthetic primers. Since primers are single-stranded DNA, they easily form complex steric structures, leading to multiple or no bands on an agarose gel. Therefore, denaturing PAGE electrophoresis is the recommended method.

Question 20

Why do modified oligonucleotides have lower yields and higher costs than standard oligonucleotides?

Accepted Answer

Modified oligonucleotides generally exhibit lower yields and higher production costs due to several inherent factors:(1) Reduced synthesis efficiency: Modified phosphoramidites are often less chemically stable and exhibit lower coupling efficiency during synthesis, resulting in decreased overall yield compared to standard oligonucleotides.(2) More stringent purification requirements: To ensure performance and purity, modified oligonucleotides typically require advanced purification methods such as PAGE or HPLC. These processes lead to additional material loss during purification.(3) Higher raw material costs: Specialized modified monomers and reagents are significantly more expensive - often by orders of magnitude - than standard synthesis materials.Together, these factors contribute to the higher cost structure and reduced yield commonly associated with modified oligonucleotides.

Question 21

Are synthesized oligonucleotides phosphorylated at the 5' end by default?

Accepted Answer

No. By default, synthesized oligonucleotides are delivered with a 5' hydroxyl (-OH) group rather than a phosphate. If phosphorylation is required, two options are available:(1) Post-synthesis enzymatic phosphorylationCustomers may perform 5' phosphorylation using polynucleotide kinase (PNK) after receiving the oligonucleotides.Custom phosphorylation during synthesis(2) 5' or 3' phosphorylation can be incorporated during synthesis upon request as a value-added service.

Question 22

Why is PAGE purification not recommended for fluorescently labeled oligonucleotides?

Accepted Answer

(1) Photodamage riskUV illumination used to visualize and excise gel bands can quench fluorescence or chemically degrade dye labels, compromising assay performance.(2) Unpredictable gel migrationFluorescent dyes alter the molecular weight and charge of oligonucleotides, leading to atypical migration behavior and making accurate band identification difficult using standard size markers.For fluorescently labeled oligonucleotides, HPLC purification is strongly recommended, as it provides high purity while preserving the integrity and performance of fluorescent modifications.

Question 23

What causes low fluorescent signal in amplification curves?

Accepted Answer

Low signal values are mostly due to high background fluorescence (for example, check the baseline-corrected or ROX-corrected raw curves). In probe-based detection, this is often caused by:        * Poor probe design: Leading to insufficient quenching by the quencher group.* Improper pairing: Incorrect matching of the reporter and quencher groups.* Low labeling efficiency: The probe's fluorescence labeling efficiency is too low.

Question 24

What causes erratic peaks in melting curves?

Accepted Answer

Erratic peaks often result from contamination in the reaction system.   * Confirm contamination using No Template Control (NTC) and No Reverse Transcriptase Control (NRC) results.* Check potential sources such as water, oligonucleotides, enzymes, and the experiment settings.   * Reagents exposed to strong light or high temperatures may degrade. It is recommended to compare results with fresh reagents.

Question 25

Why do dissolved primers perform well initially but show reduced performance after storage?

Accepted Answer

Primers that have been resuspended may degrade or produce inconsistent results over time due to several common factors:(1) Primer degradation: Resuspension in nuclease-contaminated water or solutions with non-neutral pH can lead to gradual chemical degradation or enzymatic cleavage, reducing primer integrity.(2) Improper handling: Incomplete thawing, insufficient mixing, or repeated freeze-thaw cycles may result in uneven primer concentration, causing variability in pipetting and assay performance.(3) Changes in other reaction components: Apparent primer failure may also be caused by deterioration or variation in other PCR reagents-most notably the DNA template - rather than the primer itself.Recommended Best Practices:(1) Resuspension buffer: Dissolve primers in nuclease-free 10 mM Tris-HCl (pH 7.5 - 8.0) to maximize stability.(2) Aliquot and storage: Aliquot primer solutions into single-use or low-use volumes and store at -20°C to minimize freeze–thaw cycles.(3) Thorough mixing before use: After thawing, vortex briefly and centrifuge to ensure a homogeneous solution prior to pipetting.

Question 26

Is PCR amplification failure related to the primers?

Accepted Answer

Generally not. Modern PCR techniques and high-temperature polymerases have been developed to solve most issues of amplification failure and low n efficiency. Failures are more commonly due to two main situations:(1) RT-PCR: The quality of the RNA used in the RT reaction and the content of the target gene in the tissue/cell are critical.For example, some repeat fragments, fragments with high GC content amplification, must use special amplification means to amplify.(2) Amplification from the genome: Genes are single copies in the genome, so the amount of genomic DNA template must be strictly controlled. Too much genomic DNA can affect the Mg2+ concentration and pH in the reaction system.

Question 27

Does strong non-specific banding in PCR indicate primer contamination?

Accepted Answer

No. Sequencing of non-specific fragments usually finds at least one primer sequence at both ends. Therefore non-specific amplification is typically the result of template contamination (e.g. contaminated genome in RNA) or unsuitable amplification conditions.

Question 28

What is NGS adapter?

Accepted Answer

Tsingke ensures sample uniformity by isolating monoclonal clones. Each clone is validated by Sanger sequencing, covering the entire gene and at least 50 bp of the vector backbone upstream and downstream, which ensures the gene is correct and properly placed in the vector. The plasmid then undergoes NGS to verify the accuracy of the entire plasmid and detect any contamination. Before shipment, a strict QC review is conducted.

Question 29

What are the main types of capture probes?

Accepted Answer

Capture probes are commonly classified based on their application areas, including:* Cancer research probes* Basic research probes* Hematology-related probes* Pathogen detection probes* Genetic disease probes* Methylation analysis probes

Question 30

What are the key factors to consider when designing multiplex PCR primers?

Accepted Answer

Successful multiplex PCR primer design requires careful consideration of the following factors:(1) Primer length: Primers are typically designed to be 18-24 nucleotides long to ensure specificity while minimizing the risk of primer-dimer formation.(2) Melting temperature (Tm): All primers included in a multiplex reaction should have similar Tm values to enable uniform annealing and efficient amplification under a single set of cycling conditions.(3) 3'-End complementarity: Complementarity between primers, especially at the 3' ends, should be avoided to reduce the likelihood of primer-dimer formation and non-specific amplification.(4) Stepwise optimization: Each primer pair should be optimized individually before being combined. Once pooled, primers should be mixed and further optimized incrementally to achieve balanced amplification.Adhering to these principles helps improve specificity, efficiency, and reproducibility in multiplex PCR assays.

Question 31

Why annealing temperature and cycle number are improtent in multiplex PCR?

Accepted Answer

In multiplex PCR, multiple primer pairs are used simultaneously, making optimization of reaction conditions critical for balanced and specific amplification.(1) Annealing temperature: Each primer pair has its own optimal annealing temperature. For multiplex reactions, the annealing temperature is typically set based on the lowest melting temperature (Tm) among all primers to ensure efficient binding and amplification across targets.(2) Cycle number: Using the minimum number of PCR cycles required to obtain sufficient product helps maintain amplification efficiency and specificity, while reducing nonspecific products and amplification bias between targets.Careful optimization of both annealing temperature and cycle number is essential for reliable and reproducible multiplex PCR performance.

Question 32

Why is it important to optimize reagent concentration and extension time in multiplex PCR?

Accepted Answer

In multiplex PCR, multiple targets are amplified simultaneously, increasing competition for key reagents such as DNA polymerase, dNTPs, and primers. If reagent concentrations are not properly optimized, amplification efficiency and balance between targets may be compromised.Additionally, the simultaneous synthesis of multiple amplicons increases the overall demand on the polymerase, often requiring longer extension times than single-target PCR to ensure complete and efficient synthesis of all products.Optimizing reagent concentrations and extension time is therefore essential to achieve uniform amplification, high specificity, and reliable results in multiplex PCR assays.

Question 33

What is the recommended primer length for multiplex PCR?

Accepted Answer

For multiplex PCR, primers are typically designed to be 18-24 nucleotides in length. This range provides a balance between specificity and efficient annealing, while minimizing the risk of primer-dimer formation. Longer primers are generally more prone to secondary structure formation and non-specific interactions, which can negatively affect multiplex PCR performance.

Question 34

Sequencing of cloned PCR products shows base errors within the primer region. What should I do?

Accepted Answer

This issue is most commonly associated with the inherent limitations of chemical oligonucleotide synthesis. Synthetic primers are not 100% pure, and trace amounts of truncated or failure sequences may be present. These minor impurities can occasionally participate in PCR amplification and be carried through cloning and sequencing.We recommend the following actions:(1) Sequence additional clones: Re-pick and sequence one or more additional colonies. In most cases, the initially sequenced clone originates from a PCR product amplified by a minor impurity, while other clones contain the correct sequence.(2) Contact technical support if the issue persists: If the same primer-region error is observed consistently in 2-3 independent clones, the primer batch itself may be the source. Please contact our technical support team for further assistance. We will arrange prompt resynthesis or replacement of the primer at no additional cost.

Question 35

What is the maximum length and the types of RNA oligonucleotides synthesized?

Accepted Answer

We specialize in synthesizing RNA oligos up to 180 nucleotides in length. Types mainly include: siRNA, sgRNA, miRNA, ASO, and various modified RNAs.

Question 36

How is the quality and accuracy of synthetic RNA oligonucleotides ensured?

Accepted Answer

All RNA oligonucleotides undergo rigorous quality control:(1) Mass spectrometry (MS): Every batch is verified by MS to confirm identity and mass accuracy.(2) HPLC purification: Available for applications requiring >90% purity.(3) Nuclease-free and integrity checks: Gel electrophoresis and functional validation are optional upon request.We guarantee sequence accuracy, modification integrity, and high batch-to-batch reproducibility.

Question 37

What customization options are available for custom RNA oligonucleotides?

Accepted Answer

Our custom RNA oligonucleotide services offer a high degree of flexibility, allowing you to tailor each stage of the workflow-from synthesis and quality control to packaging and delivery-to meet your specific requirements.In addition to standard RNA synthesis, we provide integrated, end-to-end solutions for small nucleic acid screening and targeted delivery. Our capabilities support high-throughput workflows and comprehensive performance evaluation, including:* Efficacy assessment: Analysis of mRNA and protein knockdown efficiency.* Stability and safety evaluation: Assessment of oligonucleotide stability, off-target effects, immunogenicity, and cytotoxicity.These services deliver reliable, highly reproducible results, enabling confident identification and optimization of lead RNA candidates with enhanced silencing performance.

Question 38

What is the most effective method for delivering oligonucleotides into cultured cells?

Accepted Answer

Several delivery strategies are commonly used, including:* Lipid-based transfection (liposomes)* Ligand conjugation (e.g., peptides, GalNAc, cholesterol, fatty acids, aptamers, antibodies)* Electroporation* Gymnotic (naked) uptakeThe optimal delivery method depends on the cell type, oligonucleotide chemistry and modifications, and the specific experimental or therapeutic application. Please contact us for technical guidance tailored to your system.

Question 39

What types of chemical modifications can be incorporated into synthetic RNA oligonucleotides?

Accepted Answer

We offer a wide range of RNA modifications:(1) Stability enhancements: 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), phosphorothioate (PS) bonds(2) Functional handles: 5'/3' amino (NH2), thiol (SH), biotin, azide, DBCO(3) Labels & detection: Fluorescent dyes (FAM, Cy3, Cy5), quenchers (BHQ)(4) Specialty bases: Pseudouridine (Ψ), 5-methylcytidineThese modifications improve stability, specificity, delivery, and functionality for applications such as RNA therapeutics and diagnostics.

Question 40

What are the maximum absorption and emission wavelengths of commonly used RNA fluorescent dyes?

Accepted Answer

Commonly used fluorescent dyes for RNA labeling have well-defined excitation (absorption) and emission maxima. Typical values are listed below:* FAM: Maximum absorption at ~495 nm; maximum emission at ~520 nm* Cy3: Maximum absorption at ~550 nm; maximum emission at ~565 nm* Cy5: Maximum absorption at ~643 nm; maximum emission at ~667 nmNote: Exact wavelengths may vary slightly depending on the dye supplier, conjugation chemistry, and measurement conditions.

Question 41

Precautions for using siRNA

Accepted Answer

(1) The product is shipped in the form of lyophilized powder, the dry powder is attached to the wall of the tube and is easily dispersed when opened, so please centrifuge the tube for a few seconds before dilution to make the product gather at the bottom of the tube and open the cap carefully, cover the tube after dissolution, vortex and shake to mix to make full dissolution. It is recommended to store at -20°C or below, avoid repeated freezing and thawing, and keep in separate containers. Centrifuge instantly before use and prepare 20~100 μM storage solution with RNase-free Water or ddH2O, the average molecular weight of siRNA is 13300.(2) RNA handling rules should be strictly followed and RNase-free lab supplies should be used for experimental operations to avoid degradation of RNA. Please keep on ice when using this product.(3) For RNA with fluorescent marker, we use brown centrifuge tube to pack, because the fluorescent marker is sensitive to light, it must be stored away from light.

Question 42

How should siRNA be handled, used, and stored?

Accepted Answer

To ensure optimal performance and stability of siRNA products, please follow the guidelines below:1. Reconstitution and StoragesiRNA is supplied as a lyophilized powder and may adhere to the tube walls during shipping. Before opening the tube, briefly centrifuge to collect the material at the bottom. Open the tube carefully to avoid sample loss.* After adding solvent, cap the tube securely and mix thoroughly by gentle vortexing to ensure complete dissolution.* Reconstitute using RNase-free water or ddH2O to a recommended stock concentration of 20-100 μM.* Store reconstituted siRNA at -20°C or below. Avoid repeated freeze-thaw cycles by aliquoting the solution into single-use portions.* Before each use, briefly centrifuge to collect the solution at the bottom of the tube.2. RNase-Free HandlingStrict RNA-handling practices must be followed. Use only RNase-free consumables and reagents to prevent degradation. During experimental procedures, keep siRNA solutions on ice whenever possible.3. Fluorescently Labeled siRNAFluorescently labeled siRNA is packaged in light-protective (amber) tubes. Because fluorescent dyes are light-sensitive, samples must be protected from light during storage and handling.Following these recommendations will help maintain siRNA integrity and ensure consistent experimental results.

Question 43

What roles do positive and negative controls play in RNAi experiments, and how should I select?

Accepted Answer

Positive controls: These are verified siRNAs targeting housekeeping or reporter genes (e.g., GAPDH, ACTB, GFP/EGFP). They are used to assess the feasibility of the entire experimental system (transfection, RNA extraction, QC, etc.). We provide effective siRNA positive controls targeting GAPDH, ACTB, GFP/EGFP.Negative controls: These are non-specific siRNAs used to demonstrate the specificity of siRNA effects. They can be universal or scrambled sequences based on requirements.

Question 44

Should the results of negative controls be the same across different groups? What if there is significant deviation?

Accepted Answer

Yes. Negative control results are generally expected to be consistent across groups. Significant variation may indicate experimental or technical issues.However,, genes involved in stress or immune responses can be sensitive to minor environmental or handling differences, which may cause variability even in negative controls.If substantial deviations are observed, we recommend reviewing experimental conditions, sample handling, and assay reproducibility to determine whether the variation is biological in nature or the result of technical factors.

Question 45

What should I do if the silencing effect is not satisfactory?

Accepted Answer

The two most common reasons are low transfection efficiency and suboptimal siRNA sequence design.(1) Check transfection/improve efficiency: If you are using siRNA for the first time or working with a new cell line, we recommend testing and optimizing the transfection efficiency. If you have already optimized the transfection conditions and the problem persists, consider switching to a different transfection reagent or method.(2) Evaluate siRNA sequence design: If the transfection efficiency is optimized but silencing effect is still poor, the sequence design may be ineffective.

Question 46

Why is no fluorescence observed after transfection?

Accepted Answer

Under normal conditions, FAM-labeled oligonucleotides emit green fluorescence (excitation ~495 nm) and Cy3-labeled oligonucleotides emit red fluorescence (excitation ~550 nm). If no fluorescence is detected after transfection, please consider the following factors:(1) Excitation settings: Verify that the microscope or imaging system is set to the correct excitation and emission wavelengths (FAM: ~495/520 nm; Cy3: ~550/565 nm).(2) Post-transfection washing: Cells should be washed promptly after transfection. Delays may reduce fluorescence intensity due to dye diffusion or degradation.(3) Storage and handling: Improper storage or prolonged light exposure can lead to siRNA degradation or fluorophore quenching. Fluorescently labeled oligonucleotides, especially FAM, must be protected from light and stored as recommended.(4) Medium replacement: Ensure that fresh culture medium is replaced in a timely manner after transfection to maintain cell health and signal stability.

Question 47

Why is there a lot of cell death after transfection?

Accepted Answer

This can be due to:* Poor cell health: Ensure cells are in optimal growth condition.* Cytotoxicity: High concentrations of siRNA or transfection reagents can be cytotoxic; adjust concentrations or switch to a less toxic reagent.* Purity: Low-purity siRNA may contain impurities that affect cell viability.* Transfection time: Prolonged transfection times can exacerbate cell death.

Question 48

Why is there low efficiency of cell transfection?

Accepted Answer

Low transfection efficiency can result from several common factors:(1) siRNA quality: Impurities or RNase contamination can reduce activity. Always use high-purity siRNA and RNase-free (e.g., DEPC-treated) consumables and reagents.(2) Complex concentration: Suboptimal ratios or concentrations of the siRNA-transfection reagent complex may limit uptake. Optimization is recommended.(3) Serum interference: Serum can inhibit complex formation. Prepare the transfection mixture in serum-free medium.(4) Antibiotics in medium: Antibiotics may negatively affect transfection efficiency and should be omitted during transfection.

Question 49

How long does siRNA work within cells? When is the best time to perform detection?

Accepted Answer

siRNA-mediated RNA interference is transient. Gene silencing typically persists for several days, and analyses are recommended within 3-4 days post-transfection.

Optimal detection window:

* mRNA levels: 24-48 hours post-transfection

* Protein levels: 48-72 hours post-transfection

The exact timing may vary depending on the target gene and cell type.

Question 50

Is transfection efficiency related to the siRNA sequence?

Accepted Answer

Transfection efficiency is primarily determined by the cell type and the transfection method, rather than the siRNA sequence itself. As a result, efficiency can vary significantly between different cell lines, even when using the same siRNA.

Question 51

Is a gene knockdown efficiency of ~60% considered unacceptable?

Accepted Answer

No. While our siRNA products are typically designed to achieve ~70% knockdown, an interference efficiency of ~60% is still considered biologically meaningful. Many published studies accept this level of knockdown, especially if it results in clear phenotypic or functional changes in the cells. In most cases, knockdown efficiency can be further improved by optimizing transfection conditions and the overall experimental setup.

Question 52

Why is it important to emphasize mRNA level detection? Can we directly detect protein and function?

Accepted Answer

siRNA acts directly on mRNA, making mRNA level detection the most direct detection indicator of its effect. While clients often want to see a corresponding drop in protein, an mRNA decline level does not always correlate exactly with the protein decline level. Possible reasons include:(1) Assay time: The mRNA decline may not yet be reflected in the protein change, or the change may not be sufficient for detection. Protein and functional assays should be pushed back slightly.(2) Cellular balance: Gene expression in cells is complex. The down-regulation of mRNA level is not exactly positively correlated with the down-regulation of protein level.

Question 53

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

Accepted Answer

Cells differ in transfection efficiency, endogenous gene expression levels, and other cellular factors that influence siRNA activity. As a result, the same siRNA may not produce identical knockdown effects across different cell types.

Question 54

What modifications are available on an HPLC purified sgRNA?

Accepted Answer

HPLC-purified sgRNAs support the same modifications as standard desalt preparations. These include common guide RNA modifications such as RNA, 2'-O-Methyl bases, and phosphorothioate linkages as well as newer options like 2'-fluoro and DNA base substitutions, within typical complexity limits.

Question 55

What is the difference between chemically synthesized sgRNA and in vitro transcribed (IVT) sgRNA?

Accepted Answer

Chemically synthesized sgRNA offers several key advantages:(1) Higher editing efficiency: Studies show chemical synthesis achieves higher editing efficiency, compared to IVT sgRNA.(2) Lower cytotoxicity: IVT sgRNA carries a 5'-triphosphate modification, which can trigger innate immune responses and cytotoxicity. Chemically synthesized sgRNA avoids theses off-target immunostimulatory effects.(3) Enhanced stability: Chemical synthesis allows the incorporation of stabilizing modifications (e.g., phosphorothioate (PS) bonds and methoxy groups). Moreover, the chemical synthesis process ensures higher batch-to-batch consistency and reproducibility.(4) Ease of use: Chemically synthesized sgRNA is ready to use upon delivery, while IVT sgRNA requires a multi-step, 2-3 day production process that , incurs greater time and economic costs.

Question 56

What is the difference between using synthetic sgRNA and the crRNA:tracrRNA duplex?

Accepted Answer

Using synthetic sgRNA eliminates the need for annealing crRNA and tracrRNA before application. More importantly, studies suggest that long single-stranded sgRNA exhibits greater stability and higher genome-editing efficiency when complexed with Cas9 compared to the crRNA:tracrRNA duplex.

Question 57

What types of modifications are routinely used in antisense experiments?

Accepted Answer

The most commonly used modification in antisense oligonucleotides is the phosphorothioate (PS) backbone, which enhances resistance to nuclease degradation.A full PS backbone is often applied to improve stability, extend circulation time, and enhance cellular uptake, particularly in the absence of delivery agents. However, PS modification may slightly reduce melting temperature (Tm) and increase non-specific protein binding, which in some cases can contribute to immune-related effects.

Question 58

What is the recommended transfection concentration for ASO?

Accepted Answer

The recommended starting transfection concentration for ASOs is 50 nM, with target gene analysis typically performed 24-72 hours post-transfection. In practice, the optimal concentration usually falls within the 10-100 nM range and should be determined by dose-response (gradient) optimization based on the specific cell line and ASO chemistry.

Question 59

How can I improve the silencing efficiency of ASO?

Accepted Answer

ASO silencing efficiency can be enhanced by optimizing several key factors:(1) Improve transfection efficiency: Optimize the transfection reagent, conditions, and cell type to maximize ASO uptake.(2) Optimize ASO concentration: Perform dose-response studies to identify the most effective concentration with minimal toxicity.(3) Redesign the ASO sequence: If target RNA secondary structures limit access, alternative target sites or sequences may significantly improve silencing performance.

Question 60

Which is the best way to deliver oligos into cells in culture?

Accepted Answer

There are several different ways to introduce oligos into cells, including use of liposomes, attaching the oligo to a ligand (e.g. peptides, GalNAc, cholesterol, fatty acids, aptamers, antibodies etc.), electroporation, and gymnotic (naked) delivery.The best method depends on the type of cells and types of modifications on the oligo, as well as the intended use of the ASO. Contact us for more information.

Question 61

What are the methods of purification?

Accepted Answer

The most commonly used purification methods are PAGE and HPLC.

Question 62

Does the type of modification affect oligonucleotide stability or shelf life?

Accepted Answer

For most commonly used modifications, overall stability and degradation profiles are comparable to those of unmodified oligonucleotides. However, comprehensive long-term stability data are not available for every modification.Recommended storage practices:(1) Store oligonucleotides in lyophilized form at -20°C for maximum shelf life.(2) After reconstitution, use TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5-8.0) rather than water to improve stability, particularly at higher temperatures.(3) Protect from light at all times and avoid UV exposure, especially for light-sensitive modifications.

Question 63

Why is the yield of modified primers lower and the price higher than that of general primers?

Accepted Answer

(1) Stability and Efficiency: Modified monomers are less stable and have longer coupling times, resulting in lower efficiency and yield.(2) Purification: They require intensive purification methods like PAGE or HPLC, leading to significant material losses.(3) Raw Material Cost: The raw materials for modified primers are much more expensive, often hundreds of times more than those for general primers.

Question 64

What are the most commonly used fluorescent dyes?

Accepted Answer

Commonly used fluorescent dyes include FAM, TET, HEX, TAMRA, ROX, Cy3, and Cy5. These dyes cover a broad range of excitation and emission wavelengths and are widely applied in PCR, hybridization assays, and fluorescence-based detection.

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