Silencer™ siRNA Construction Kit, 15 reactions - FAQs

Vector technologies allow you to:

Achieve transient or stable target knockdown
Perform RNAi in any cell type, even hard-to-transfect, primary, and non-dividing cells
Regulate gene inhibition with inducible siRNA expression
Select for a pure population of cells stably expressing an siRNA sequence
Control gene expression in vivo with tissue-specific promoters

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We would suggest running a transfection reagent control only to determine if your cells are sensitive to the transfection reagent. Additionally, you can try using different cell densities and siRNA concentrations to diminish any toxic effects from the transfection itself.

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In some cases, knockdown of a protein can be affected by other variables such as protein turnover rate, even though the RNA is knocked down. Additionally, a longer time course may be needed to see an effect on protein compared to mRNA.

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Please see the following possibilities and suggestions:

- How many siRNA did you test? Is there any knockdown? If there is no knockdown (<10%) in any of the siRNA, then the assay is likely the problem. Try using a different qRT-PCR assay to assess knockdown.
- What was the positioning of the qRT-PCR assay target site relative to the cut site for the siRNA? If greater than 3,000 bases away, the problem could be alternative splice transcripts.
- What are the Cts for the experiment? They should be below 35 in a 40-cycle qRT-PCR experiment.
- Did you confirm the siRNA got into the cell? We recommend using a validated positive control siRNA to check the transfection efficiency.

Find additional tips, troubleshooting help, and resources within our RNAi Support Center.

Please see the following possibilities and suggestions:

- Were the mRNA levels checked? The most reliable method is real-time PCR. In some cases, knockdown of a protein can be affected by other variables, such as protein turnover rate, even though the RNA is knocked down.
- How is the RNA being isolated? Has the quality of the isolated RNA been checked? Ensure that the RNA has not been degraded.
- Was a positive control used? This can help to determine whether the reagents are working and whether the siRNA was delivered correctly to the cell. Run your experiment in parallel with the positive control siRNA.
- Was a transfection control used? What is the percentage of transfected cells?
- Was a time course used? Generally, gene silencing can be assessed as early as 24 hours posttransfection. However, the duration and level of knockdown are dependent on cell type and concentration of siRNA.
- Was optimization of transfection conditions performed? You can try using different cell densities and siRNA concentrations.
- Which concentration of siRNA did you use? We recommend testing multiple concentrations between 5 nM and 100 nM.

Find additional tips, troubleshooting help, and resources within our RNAi Support Center.

Depending on the gene you are working with, it can be measured at the mRNA level as soon as a few hours after transfection. We recommend assessing the mRNA knockdown 48 hours posttransfection. Factors affect the timing include the transcription activity, the turnover rate for the mRNA, and if there are alternative pathways. To determine the peak knockdown, it is best to perform a time course experiment.

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We recommend dissolving the single stranded RNA in 1X TE buffer (prepared under RNase-free conditions (10 mM TrisCl, pH 7.5, 0.1 mM EDTA). This buffers the pH and chelates metal ions that can contribute to RNA degradation. RNase-free water is also acceptable. Duplex RNA (siRNA) comes lyophilized from 10 mM Tris-HCl, pH 8.0, 20 mM NaCl, 1 mM EDTA. Resuspending in the appropriate amount of nuclease free water to bring the RNA conc. to 20 µM will reconstitute the buffer to the same.

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As a dry pellet they can be stored at -20 degrees C for 6 months.

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Measure mRNA levels by real-time PCR. A typical time course analysis would measure at 24, 48, and 72 hours. Protein analysis may be checked at 48, 72, and 96 hours. This will vary depending on the gene being targeted.

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We suggest using at least 2 siRNA targeting the same gene. This will give greater confidence in RNAi data.

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Custom Silencer or Silencer Select siRNAs (21 bases in length) can be ordered using the following link: https://www.thermofisher.com/order/custom-genomic-products/tools/sirna/

For longer or shorter siRNA, please email oligos@invitrogen.com.

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Pre-designed Silencer siRNA are algorithm designed and have not been functionally tested. They are available for human, mouse and rat genes and designed to target all known splice variants. The sequence information is provided once the siRNA is purchased. We guarantee 2 out of 3 Silencer and 2 out of 2 Silencer Select siRNA targeted to the same gene will give you 70% or greater knockdown. Validated siRNA are algorithm-designed siRNA that have been experimentally verified to knock down mRNA levels by 70% or more. They are individually guaranteed to silence, and available for select human genes. Validation data is available, and sequence information is provided once siRNA is purchased.

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Silencer siRNAs were our first-generation siRNAs, while Silencer Select siRNAs were our second generation of Invitrogen siRNAs. They were designed using different algorithms, but both contain a 19-nucleotide core sequence plus a 2-nucleotide 3' overhang. Silencer Select siRNAs also contain a chemical modification. We recommend using Silencer Select siRNA whenever possible, as they show enhanced efficacy, specificity, and potency.

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In order to analyze the effects of a specific siRNA sequence on gene activity, the introduced siRNA sequence must be known. This requires the design, introduction, and measurement of gene blocking following the addition of synthetic siRNA oligonucleotides or of a short hairpin RNA (shRNA) sequence in a vector. When diced siRNAs (d-siRNA) are introduced into the cell, they are particularly effective at initiating an RNAi effect because generally there will be several effective siRNA sequences that are part of the pool. However, there currently there is no way to identify the specific sequence(s) in a pool that are responsible for the effect.

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The most common way to measure gene specific knockdown is to perform real-time PCR. In some cases a reporter system that allows easy measurement of a reporter gene, such as beta-galactosidase, may be used. Western blot analysis to compare the level of protein expression before and after the introduction of siRNA may also be employed.

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The structure of the molecules is the same: Dicer specifically cleaves long dsRNA into the 21-23 nucleotide duplexes with a 2-nucleotide overhang that is the hallmark of siRNA. A key difference is that d-siRNA typically contains a pool of siRNA generated from the entire length of a long dsRNA target, whereas siRNA generally refers to a single sequence that is specific to a particular target region, and is often synthesized as a single oligo or a specified combination of several oligos.

Find additional tips, troubleshooting help, and resources within our RNAi Support Center.

The three most common methods of generating siRNA to introduce into mammalian cells are:

- In vitro transcription and dicing
- Synthetic siRNA
- Vectors carrying an RNAi cassette

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RNAi is a cost-effective method for the rapid identification of gene function, and appears to work well for most genes tested to date. RNAi is rapidly becoming the preferred method for knocking out the expression of targeted genes. RNAi is useful for assigning gene function, signaling pathway analysis, RNAi mechanism studies, target validation, and shows tremendous potential for diagnostics and therapeutics.

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Custom Designed: If a pre-designed siRNA is unavailable, we will use our algorithm to custom design one for you using the target mRNA's nucleotide sequence or accession number (transcript variants, species other than human, mouse, rat). Custom designed siRNAs are not guaranteed.
Custom Synthesized: The customer provides the siRNA sequence, and we synthesize it. Custom synthesized siRNAs are not guaranteed.
Custom Modified: A quote can be requested for custom modifications such as fluorescent labels (FAM, Cy3), custom sizes ( greater than 50 nmol) and aliquotting. Custom modified siRNAs are not guaranteed.

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Off-targeting effects are when any gene-silencing effects are caused by siRNAs on nontarget mRNAs through the RNAi mechanism. They could come from the guide (antisense) or passenger (sense) strand of siRNA.

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Typically, we would recommend matching the experimental and control siRNAs by brand. There are differences in siRNA length and modification between Silencer, Stealth, and Silencer Select sequences. A good experimental design would minimize these variables between experimental and control siRNA. That said, in a bind, using a negative control with a different brand than the experimental siRNA would still help to control for off-target effects.

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Yes, we offer a pSCREEN-iT/lacZ-DEST Gateway Vector Kit which can be used to assess the potency of any RNAi knockdown without knowing protein function, western blotting or qRT-PCR. The system uses a beta-galactosidase activity assay to measure the knockdown ability of an RNAi reagent (Stealth siRNA, Silencer siRNA, shRNA-containing plasmid, Dicer pools, etc). Clone your target gene into the vector provided, and co-transfect it along with the knockdown reagent being tested. Expressed genes will be fused to beta-galactosidase, enabling you to use beta-Gal levels to measure the amount of RNAi-induced degradation of the target gene.

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A negative control is meant to reveal sequence-independent effects of siRNA on your cells. It should match the general chemistry of your positive molecule (length, modification) but be made up of a nontargeting sequence that will not target any specific gene.

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Yes, we offer several choices for negative and positive controls. Negative controls are typically universal nontargeting sequences, while positive controls are reporter genes that can be used as positive controls and/or protocol validation. Please select a control that has the same modification as your siRNA (i.e., Silencer Select Negative Control, Stealth Negative Control).

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The purification method will depend on your experiment. Please see our general guidelines below:

Standard purification:
Desalted and analyzed by MALDI-TOF mass spectrometry
Guaranteed to be at least 80% full-length product
Recommended for adherent cell lines

HPLC:
HPLC purified and analyzed by MALDI-TOF and analytical HPLC
Guaranteed to be at least 97% full-length product
Recommended for electroporation of primary or suspension cell lines

In vivo-ready:
HPLC purified, analyzed by MALDI-TOF and analytical HPLC, dialyzed to remove salts, sterile filtered, and endotoxin tested
Guaranteed to be at least 97% full-length product
Recommended to researchers using siRNA in animals
Please note that, in all cases, the efficiency of annealing is analyzed by PAGE.

Find additional tips, troubleshooting help, and resources within our RNAi Support Center.

Please see the following:

(a) Silencer Select siRNA: “Thermo Fisher Scientific guarantees that when you purchase two Silencer Select Pre-designed siRNA to the same target, then those two siRNAs will silence the target mRNA by 70% or more. To qualify for the guarantee, siRNAs must have been transfected at ?5 nM and mRNA levels detected 48 hours posttransfection. Real-time RT-PCR is recommended but not required for this application. Customers must also show sufficient knockdown with a positive control siRNA to demonstrate transfection efficiency. If the guaranteed level of knockdown is not observed and an appropriate positive control is successful, a new Silencer Select siRNA sequence will be synthesized free of charge. This guarantee does not extend to any replacement product.”
(b) Stealth siRNA: “Thermo Fisher Scientific guarantees that when you purchase three Stealth Pre-designed siRNA to the same target, then at least two of those three independent, nonoverlapping siRNAs will silence the target mRNA by 70% or more. To qualify for the guarantee, siRNAs must have been transfected at ?20 nM and mRNA levels detected 48 hours posttransfection. Real-time RT-PCR is recommended but not required for this application. Customers must also show sufficient knockdown with a positive control siRNA to demonstrate transfection efficiency. If the guaranteed level of knockdown is not observed and an appropriate positive control is successful, a new Silencer siRNA sequence will be synthesized free of charge. This guarantee does not extend to any replacement product. We also recommend the use of an appropriate negative control, such as one of the three Stealth RNAi Negative Controls, to normalize message knockdown.”
(c) Silencer siRNA: “Thermo Fisher Scientific guarantees that when you purchase three Silencer Pre-designed siRNAs to the same target, at least two of the siRNAs will reduce target mRNA levels in cultured cells by 70% or more when measured 48 hours after transfection at 100 nM or higher final siRNA concentration under the conditions described below. If at least two of the three siRNAs do not induce >70% target mRNA knockdown, Thermo Fisher Scientific will provide a one-time replacement of up to three Silencer Pre-designed siRNAs per target at no additional charge. Requests for replacement product must be made within one hundred and eighty (180) days from the date of delivery of the Silencer Pre-designed siRNAs. Optimum transfection efficiency must be confirmed using good laboratory practices and a proven-to-work siRNA to an endogenous message, such as Invitrogen Silencer GAPDH siRNA Control. To assess knockdown, target mRNA levels in treated samples must be compared to that of cells transfected with a nontargeting control siRNA, such as Silencer Negative Control #1. We recommend Applied Biosystems TaqMan Gene Expression Assays to quantify mRNA levels.”

Find additional tips, troubleshooting help, and resources within our RNAi Support Center.

We recommend the following controls:

- Positive control siRNA
- Negative control siRNA
- Cells-only control
- Multiple siRNAs per target
- Transfection reagent alone


Find additional tips, troubleshooting help, and resources within our RNAi Support Center.

Yes, if the cells are doing fine with the transfection protocol.

Find additional tips, troubleshooting help, and resources within our RNAi Support Center.

Depending on the gene you are working with, it can be measured at the mRNA level as soon as a few hours after transfection. We recommend assessing the mRNA knockdown 48 hours posttransfection. Factors affecting the timing include the transcription activity, the turnover rate for the messenger, and if there are alternative pathways. To determine the peak knockdown, it is best to perform a time course experiment.

Find additional tips, troubleshooting help, and resources within our RNAi Support Center.

The antisense (guide) strand will bind to the mRNA.

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siRNA stands for “short interfering RNA”. It consists of two complementary RNA strands 19-21 nucleotides long, with TT or dTdT overhangs. Longer dsRNA will trigger increased immune responses and be degraded. The overhangs are thought to be added by dicer when the molecules are processed. Target cleavage is thought to be in the middle of anti-sense sequences, so the middle bases need to be conserved. The synthetics will be delivered into cells to initiate RNAi via electroporation or lipid delivery. Once delivered, the two strands will separate, releasing the antisense strand. With the aid of a protein, RISC, it binds to a complementary sense sequence on the molecule of mRNA. If the base-pairing is exact, the mRNA is destroyed.

Find additional tips, troubleshooting help, and resources within our RNAi Support Center.

RNAi stands for RNA interference. The molecules that mediate RNAi are short dsRNA oligonucleotides that are processed internally by an enzyme called Dicer. The Dicer cleavage products were first referred to as short interfering RNA, now known as siRNA. RNAi technology takes advantage of the cell's natural machinery to effectively knock down expression of a gene with transfected siRNA. There are several ways to induce RNAi: synthetic molecules, RNAi vectors, and in vitro dicing. In mammalian cells, short pieces of dsRNA - short interfering RNA- initiate the specific degradation of a targeted cellular mRNA. In this process, the antisense strand of siRNA becomes part of a multiprotein complex, or RNA-induced silencing complex (RISC), which then identifies the corresponding mRNA and cleaves it at a specific site. Next, this cleaved message is targeted for degradation, which ultimately results in the loss of protein expression.

Find additional tips, troubleshooting help, and resources within our RNAi Support Center.

If gel analysis reveals a single band that migrates as though it is smaller than a 21 bp dsRNA, consider the following:

(a) Gel mobility comparison is not always an accurate way to estimate siRNA size. The gel mobility of siRNA is highly dependent on its nucleotide composition. Because of this, the gel migration of siRNAs with different sequences cannot reliably be compared to determine size. A better standard to estimate the size of an siRNA is the undigested dsRNA from which it is derived.
(b) Check the size of the template oligonucleotides and siRNA transcripts: Confirm that the sense and antisense template oligonucleotides are 29 nt, and that the individual siRNA transcripts are approximately 31 nt by running them on a polyacrylamide gel (you would need to reserve an aliquot of the sense and antisense transcripts from Step 3 on page 13 of the manual (https://tools.thermofisher.com/content/sfs/manuals/1620ME.pdf) before combining them). A shorter-than-expected template oligonucleotide or a sequence in the template that disrupts T7 RNA polymerase could create smaller than expected siRNAs. If the template oligonucleotide is smaller than expected, then have it resynthesized. If the template is the correct size but either or both of the transcripts is smaller than expected, then design oligonucleotides to a different target sequence.
(c) Confirm that the sense and antisense oligonucleotides are entirely complementary: A single base mismatch could create a cleavage site for the RNase used in the digestion reaction, resulting in a smaller than expected siRNA.

Find additional tips, troubleshooting help, and resources within our RNAi Support Center.