Silencer™ Select Human GPCR siRNA Library V4 - 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.

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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.

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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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Yes, most Silencer and Silencer Select siRNAs have dTdT overhangs on the sense strand. The important thing is that the antisense (guide strand) 3' overhang will be complementary to the 5' end of the target mRNA. However, the overhangs could have any nucleotide composition.

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Yes, we have a pre-plated collection available for the entire human genome and for different functional classes as Silencer Select Libraries. We also offer custom Silencer Select library services and/or Silencer Libraries for human and mouse.

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The average molecular weight of the Silencer Select siRNA is 13,400 g/mol where 1nmol = 13.4 µg.

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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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The modification is LNA - locked nucleic acid- which improves thermal stability and specificity of duplexes formed with complementary RNA. The location of this modification is proprietary.

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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.

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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.

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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.”

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We recommend the following controls:

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


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

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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.

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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.

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The goal of transfection optimization is to determine the conditions that will provide maximum gene knockdown while maintaining cell viability for the particular cell type. There is no single transfection parameter that by itself ensures efficient uptake by cells in culture. However, there are several critical variables to consider in systematically addressing the optimal siRNA uptake into cells:

• Health of cultured cells
• Cell type
• Transfection method
• Choice and volume of transfection agent
• Exposure time of cells to transfection agent
• Cell density
• Amount of siRNA

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Transfection efficiency achieved using transfection delivery strategies depends on the type of cell being transfected. Cells types differ in features that are associated with high or low siRNA delivery efficiency and viability. Easy-to-transfect cells are those that are readily transfected by traditional lipid or electroporation methods and result in high delivery of siRNA and cell viability. These are typically adherent cells and have flat growth morphology. Difficult to transfect cells are cell types that are intractable to traditional lipid or electroporation strategies. For example, suspension, primary, neuronal, and stem cells are historically difficult to transfect. In addition, difficult-to-transfect cells may have clustered growth morphology.

We generally don't recommend pooling. However, it is up to the scientist to design the experiment that fits their needs best. The major justification for pooling siRNAs for screening is that it reduces off-target effects. While this was a major issue with first-generation siRNAs that were not chemically modified and may have had targeting 3'UTR and acted like miRNA, it is not an issue with newer generations of siRNAs such as Silencer Select siRNAs. Since Silencer Select siRNAs are designed using advanced bioinformatic algorithms and rules, and also incorporate chemical modification, one can achieve robust and guaranteed KD at low concentration with very minimal off-target effects. Pooling involves de-convolution of siRNAs for repeat or second screening. Also, siRNA pooling increases the chance of false positive hits that correlate poorly with subsequent single siRNA screening results.

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This is cell line and reagent dependent but we have found that this complex is stable. We tested up to 3 hrs and there was no decline in knockdown in HeLa cells.

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This is cell line and reagent dependent but we have found that diluted RNAiMAX reagent is quite stable. We tested up to 2 hrs following transfections into HeLa cells with good knockdown. However, we noticed a difference if the lipid was diluted in a tissue culture plate versus a conical tube, but if the transfections occur within an hour there isn't a problem.

We have pre-plated Silencer Select siRNAs (1, 30, 50 and 100 nM final concentration) and left the plates out at room temperature overnight. These plates were then transfected with RNAiMAX reagent in HeLa cells the next day and greater than 80% knockdown of our target CSNK2A1 was still achieved.

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The optimal transfection agent depends upon the cell type used. That being said, we find that Lipofectamine RNAiMAX transfection agent provides excellent transfection efficiency with low toxicity in most cell types.

Good transfection is absolutely essential for effective target knockdown using siRNA, thus it is important to include a positive control siRNA in each experiment. The positive control siRNA should elicit reproducible, easily measured knockdown and/or phenotype in the cells and assay used in your study. If you see maximal knockdown or a phenotype above/below a pre-determined threshold level with this control, you know that measurements from other siRNAs tested on the same day are reliable. Note that it is important to empirically determine the thresholds for each assay and siRNA control pair that indicate a good transfection.

In siRNA experiments, negative controls are just as important as positive controls for obtaining meaningful data. Always include a set of transfections with an equimolar amount of at least one non-targeting negative control siRNA (e.g., Invitrogen Silencer Select Negative Control #1) - data from these crucial controls serve as a baseline for evaluation of experimental target knockdown.

Nontransfected or cells-only negative controls are also very useful in siRNA experiments. By comparing expression of a housekeeping gene among cultures that were not transfected and cultures transfected with a non-targeting negative control siRNA, valuable information about the effects of transfection on cell viability can be obtained.

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Identity: the mass of a sample of each single-stranded RNA oligonucleotide is analyzed using MALDI-TOF mass spectrometry and compared to the calculated mass.
Annealing: a sample of the annealed siRNA is analyzed by nondenaturing gel electrophoresis.

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Even after resuspension in water, stock solutions of Silencer Select siRNAs are remarkably stable to freeze-thaw cycles. We have tested Silencer Select siRNAs at 1, 5, 10, and 100 µM concentrations after 100 freeze thaw cycles, and have seen no loss of siRNA integrity when analyzed by HPLC and no loss in function when transfected into cells at 5 nM with knockdown measured two days later by qRT-PCR.

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Invitrogen siRNA Libraries are dried and are therefore shipped at ambient temperature. Although dried siRNAs are remarkably stable, we recommend that you store the dried siRNAs at -20 degrees C (or lower) until ready for use.

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The siRNAs can be stored at 4 degrees C for short-term uses but care should be taken to seal well to avoid evaporation.

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The working dilution plates should be at a concentration that will allow accurate pipetting into the transfection plates. For most screening situations, we find that 100 nM siRNA is a convenient working solution concentration. For 96- well plates, 0.5 pmol siRNA per well in 100 µl final transfection volume gives a 5 nM final siRNA concentration. Therefore, you would use 5 µl of the 100 nM siRNA working dilution.

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For those researchers who want to re-seal plates after resuspension, we recommend aluminum seals over plastic. We have used the following seal in the past with good success:

Catalog Number: 47734-816
Supplier: Axygen Scientific
Description: Aluminum Sealing Film. Maintains excellent seal on microplates under the widest temperature conditions (-80 degrees C to 97 degrees C) and reagents.

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Store at -20 degrees C or lower for long-term storage. The siRNAs can be stored at 4 degrees C for short-term uses but care should be taken to seal well to avoid evaporation.

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Plates are shipped at ambient temperature. In the US and Canada, we use FedEx to ship these products.

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We use a heat seal system to affix the seals on Invitrogen siRNA Library plates. To prevent the glue sticking to the plate, you may want to remove the seal while the plate is still cold.

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Yes. Please note that these lids are not labeled, so you do not need to worry about trying to keep lids and plates aligned.

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We use Axygen clear 96-well full-skirt PCR microplates. (Cat. No. PCR-96-FS-C; we use sterile versions of this plate.)

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The following human Silencer Select siRNA libraries are available:

Genome
Extended druggable genome
Druggable genome
Kinases
Phosphatases
GPCRs
Ion channels
Nuclear hormone receptors
Proteases

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If you still would like to have 4 siRNAs in your library instead of 3, you have the option of adding 1 siRNA from our Silencer designs for the human library.

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It is generally agreed that you need at least two effective siRNA per target to confirm that a phenotype observed is due to knocking down the intended gene and not due to an off-target effect. Due to the strength of the Silencer Select siRNA design algorithm and the novel chemical modification, which serve to reduce off-target effects without negatively impacting siRNA potency, a significantly higher percentage of Silencer Select siRNAs are effective at eliciting on-target phenotypes as compared to other siRNA technologies.

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Turnaround times vary depending on the number of siRNAs and the complexity of the plating request. Most Invitrogen siRNA library orders are completed within 1-3 weeks of order processing. An estimated turnaround time for your particular library will be provided with your price quote. Please contact your local sales representative or email us at rnailibraries@lifetech.com for more information or a quote.

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For custom siRNA libraries, we can provide a number of plating options. Our most requested format is to have the last 1 or 2 columns empty of each 96-well plate, and to have different siRNAs to the same targets plated in separate plates in the same well location. The empty columns facilitate easy inclusion of any desired control in the final transfection plates. When different siRNAs to the same targets are plated in separate plates in the same well location, the siRNAs can easily be pooled with robotics or a multichannel pipettor. In addition, this type of format facilitates analysis by qRT-PCR.

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Your gene list and custom siRNA information is kept strictly confidential and is not used for any other purpose other than to prepare your price quote or to manufacture your custom siRNA library.

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The Silencer and Silencer Select siRNA Human Genome siRNA Library collections include three siRNAs for most human coding genes listed in the NCBI Entrez Gene database. We generally keep these siRNAs in stock, and therefore, our costs and your prices are lower for these siRNAs than ones that we custom manufacture just for you. To provide a quote, we must compare your gene list to our list of inventoried siRNAs.

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Contact your local sales representative or contact us at rnailibraries@lifetech.com. We will provide you with a Quote Request form to indicate exactly how you would like your siRNA library formatted. You will need to be prepared to provide the following information:

Your gene/target list (preferably by NCBI Entrez Gene ID; RefSeq mRNA Accession Number is also acceptable)
Your choice of Silencer Select siRNA, Silencer siRNA (unmodified), or Invitrogen In Vivo siRNA
The number of siRNAs needed per target (typically 3 for coding genes)
The amount of siRNA per well desired:
Silencer Select siRNAs available as 0.1, 0.25, 1, 2, or 5 nmol
Silencer siRNAs available as 1, 2, or 5 nmol
Your desired plating format

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Below are some citations that reference Invitrogen siRNA libraries/collections. This is only a subset. Please contact us for an updated citation list at rnailibraries@lifetech.com.

Silencer Select siRNA
Phenotypic profiling of the human genome reveals gene products involved in plasma membrane targeting of Src kinases. Ritzerfeld J, Remmele S, Wang T et al. (2011) Genome Res Jul 27 (available online)
Identification of protein kinases that control ovarian hormone release by selective siRNAs. Sirotkin AV, Ovcharenko D, Mlyncek M (2010) J Mol Endocrinol 44:45-53.
Selection of hyperfunctional siRNAs with improved potency and specificity. Wang X, Xiaohui Wang X, Varma RK et al. (2009) Nucleic Acids Res:37: e152.
Protein kinases controlling PCNA and p53 expression in human ovarian cells. Alexander V Sirotkin AV, Ovcharenko D, Benco A et al. (2009) Funct Integr Genomics 9:185-195.

Silencer siRNA
Identification of host factors involved in borna disease virus cell entry through a small interfering RNA functional genetic screen. Clemente R, Sisman E, Aza-Blanc P et al. (2010) J Virol 84:3562-3575.
siRNA screening reveals JNK2 as an evolutionary conserved regulator of triglyceride homeostasis. Grimard V, Massier J, Richter D et al. (2008) J Lipid Res 49:2427-2440.
Polo-like kinase 2 (PLK2) phosphorylates alpha-synuclein at serine 129 in central nervous system. (2009) Inglis KJ, Chereau D, Brigham EF et al. J Biol Chem 284:2598-2602.
Identification of survival genes in human glioblastoma cells by small interfering RNA screening. Thaker NG, Zhang F, McDonald PR et al. (2009) Mol Pharmacol 76:1246-1255.
Low-dose arsenic trioxide sensitizes glucocorticoid-resistant acute lymphoblastic leukemia cells to dexamethasone via an Akt-dependent pathway. Bornhauser BC, Bonapace L, Lindholm D et al. (2007) Blood 110:2084-2091.
Visual screening and analysis for kinase-regulated membrane trafficking pathways that are involved in extensive beta-amyloid secretion. Adachi A, Kano F, Saido TC et al. (2009) Genes Cells 14:355-369.
Identification and characterization of 3-iodothyronamine intracellular transport. Ianculescu AG, Giacomini KM, Scanlan TS (2009) Endocrinology 150:1991-1999. High-throughput RNAi screening in vitro: from cell lines to primary cells. Ovcharenko D, Jarvis R, Hunicke-Smith S et al. (2005) RNA 11:985-993.

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We use a heat-seal system to affix the seals on Invitrogen siRNA library plates. To prevent the glue from sticking to the plate, you may want to remove the seal while the plate is still cold (from the -20 degrees C or -80 degrees C freezer).

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Yes. Please note that these lids are not labeled, so there are no worries about trying to keep lids and plates aligned.

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The Silencer Select Human Genome Library can be purchase in 384-well plates or 96-well plates. There are 186 384-well plates or 738 96-well plates in the respective library formats. Each 384-well plate has the last 2 columns empty, while the 96-well plate has the last column empty.

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We can provide updated annotation information by simply rerunning your siRNA library file against our database of continually updated annotation information. To receive updated annotation information for a previously purchased siRNA library, contact Thermo Fisher Scientific Technical Support at rnailibraries@lifetech.com and provide the Lot Number of your siRNA library.

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An Excel file is provided with the following columns. Each row of the file represents a single siRNA.

1. Lot Number: A unique lot number assigned to your order.
2. Plate ID: The unique plate barcode that is affixed to the plate. This barcode is also written in human readable format on the plate label.
3. Sample ID: The unique lot number of the siRNA listed on that row.
4. Plate Name: The human readable plate name as listed on the plate label.
5. Location (Row-Col): The row and column designation of the siRNA within the plate.
6. Row: The row designation of the siRNA within the plate.
7. Column: The column designation of the siRNA within the plate
8. RefSeq Ascession Number: The RefSeq accession number(s) targeted by the siRNA
9. Gene Symbol: The NCBI Entrez Gene Symbol corresponding to the RefSeq target(s)
10. Full Gene Name: The NCBI Entrez Gene Name corresponding to the RefSeq target(s)
11. Gene ID: The NCBI Entrez Gene ID corresponding to the RefSeq target(s)
12. siRNA ID: A unique identifier representing the siRNA sequence. We use Part Numbers (also called Catalog Numbers) to designate the size and purity option, and the siRNA ID to represent the siRNA sequence. To reorder an siRNA, you will need to provide both a Part Number and siRNA ID.
13. Amount: The amount of siRNA per well, in nmol.
14. Exons(s) Targeted: The exon(s) targeted by the siRNA.
15. Sense siRNA Sequence. The sequence, listed 5' to 3', of the sense (passenger) siRNA strand.
16. Antisense siRNA Sequence: The sequence, listed 5' to 3', of the antisense (guide) siRNA strand.
17. Validated: A - Yes, in this column indicates that we have validated this particular siRNA sequence in house by qRT-PCR and that it has passed our knockdown criteria (80% or better mRNA knockdown at 5 nM for Silencer Select siRNAs).
18. Mean RNA Levels Remaining: The percent of mRNA remaining found in the validation experiments (if applicable).
19. Plus Error Bar: The upper error bar of the Mean RNA Levels Remaining (if applicable).
20. Minus Error Bar: The lower error bar of the Mean RNA Levels Remaining (if applicable).
21. Cell Line: The cell line used for siRNA validation (if applicable).

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The plate names are meant to be used as the human readable plate identifiers and typically end in a combination of letters and numbers that can be used to identify plates of siRNAs to the same targets. All of our stocked libraries, and many custom libraries, are plated with different siRNAs to the same targets provided in separate plates in the same well location. This can be thought of as - ABC? format, as the - A siRNA designs within a plate correspond to - B? siRNA designs to the same targets on separate plates in the same order.

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Yes, each plate of the library is provided with a unique barcode. No two plates have the same barcode identifier, and we can trace back samples on any plate given that barcode. The barcode identifiers are always listed in the electronic data file shipped with each library.
The barcode is located on the short end of the plate by column 12 (centered). This label includes the barcode, a human readable definition of that barcode and a plate name that helps users order plates within a library.

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The Silencer Select Human Genome siRNA Library and its subsets (Silencer Select Human Druggable Genome, Silencer Select Human Druggable Genome Extension Set, and Silencer Select Human Genome Extension Set) are plated in 384-well plates, each at 0.25 nmol siRNA per well. Each plate has the last 2 columns empty, and siRNAs to the same target are plated in different plates in the same well location. All other premade Invitrogen siRNA libraries are plated at 0.25 nmol siRNA per well in 96-well plates. Each plate has the last column empty, and siRNAs to the same target are plated in different plates in the same well location.

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2.8% of sequences in the Silencer Select Human Genome siRNA Library V4 are identical to those in the Silencer Human Genome siRNA Library V3.

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The Silencer Select Human Druggable Genome siRNA V4 siRNA Library targets 9,032 genes, whereas the Silencer Select Human Extended Druggable Genome siRNA V4 siRNA Library targets those genes plus the "Extension Set", for a total of 10,415 genes, including transcription factors.

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The Silencer Select Human Genome siRNA Library V4 (Cat. No. 4397926) contains the following:

- Silencer Select Human Druggable Genome siRNA Library V4, 384-well plates (Cat. No. 4397922). This library contains 27,093 unique siRNAs (0.25 nmol) targeting transcripts from each of 9,031 human genes. Total of 78 plates: 75 plates with 352 siRNAs each, 3 plates with 232 siRNAs each.
- Silencer Select Human Druggable Genome siRNA Extension Set V4, 384-well plates (Cat. No. 4397924). This library contains 4,149 unique siRNAs (0.25 nmol) targeting transcripts from each of 1,383 human genes. Total of 12 plates: 9 plates with 352 siRNAs each, 3 plates with 327 siRNAs each.
- Silencer Select Human Genome siRNA Extension Set V4, 384-well plates (Cat. No. 4397923). This library contains 33,510 unique siRNAs (0.25 nmol) targeting transcripts from each of 11,170 human genes. Total of 96 plates: 93 plates with 352 siRNAs each, 3 plates with 258 siRNAs each.
A list of targets is available upon request through rnailibraries@lifetech.com.

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siRNAs in the Silencer Select Human Genome siRNA Library V4 correspond to each of 21,584 genes, with 3 unique, nonoverlapping siRNAs provided per target, for a total of 64,752 siRNAs. The so called target genes correspond to greater than 98% of genes listed by NCBI that have at least one or more curated RefSeq coding transcripts. The siRNAs were designed to hit all RefSeq coding transcripts of that gene that were known at the time of design. The siRNAs targeting the druggable portion of this library are arranged by gene functional class to enable easy screening of important gene subsets.

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The following human Silencer Select siRNA libraries are available off the shelf:

- Genome
- Extended Druggable Genome
- Druggable Genome Kinases
- Phosphatases
- GPCRs
- Ion Channels
- Nuclear Hormone Receptors
- Proteases

We also provide the following predefined Silencer Select siRNA libraries as made-to-order from inventory:
- Cancer Genome
- Transcription Factors
- DNA Damage Response
- Apoptosis Response
- Epigenetics
- Drug Targets
- Drug Transporters
- Cell Cycle Regulation
- Membrane trafficking Transporter
- Ubiquitin Conjugation
- Tumor Suppressor Cell Surface

For our Silencer siRNA libraries, we offer human and mouse pre-defined sets for:
- Druggable Genome
- Genome
- Kinase
- Phosphatase

We also provide the following human Silencer siRNA libraries:
- G protein coupled receptors
- Human proteases, ion channels and nuclear hormone receptors

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Efficient and reproducible transfection is critical for any siRNA experiment. Success or failure of a siRNA experiment often hinges on siRNA delivery. Optimizing siRNA delivery and cell viability during transfection eliminates the most common causes of unsuccessful gene silencing experiments. Optimal transfection conditions vary depending on cell line. As a result, it is imperative to optimize transfection conditions for your cells before screening a siRNA library to empirically determine the highest levels of gene silencing while minimizing toxicity associated with the transfection process.

Investing the time up front to identify the best transfection conditions for your experimental system will limit transfection variability and maximize data quality by maximizing siRNA uptake and minimizing cell toxicity. Once you have identified an optimized transfection protocol, the interesting and productive work of library screening can begin.

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Most researchers find it inconvenient to cherry-pick siRNAs from their initial siRNA library plates to obtain siRNAs that they want to retest or otherwise follow up on. All of our stocked libraries, and many custom libraries, are plated with different siRNAs to the same targets provided in separate plates in the same well location. This makes it very convenient to collect siRNAs for each target easily for follow-up experiments. Also, Thermo Fisher Scientific can readily provide you with cost-effective follow-up siRNAs for your screen and provide identical siRNAs, or additional siRNAs to the same target, in either plates or tubes. Contact us for pricing and turnaround time information.

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We have pre-plated Silencer Select siRNAs (1, 30, 50, and 100 nM final concentration) and left the plates out at room temperature overnight. These plates were then transfected with Lipofectamine RNAiMAX reagent in HeLa cells the next day, and greater than 80% knockdown of our target CSNK2A1 was still achieved. Customers will need to optimize for their cell line, as the level of knockdown will differ.

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The efficiency with which mammalian cells are transfected with siRNA will vary according to cell type and the transfection agent used. This means that the optimal concentration used for transfections should be determined empirically. Since Silencer Select siRNAs exhibit superior silencing potency compared to other siRNAs, we suggest starting concentrations of 5- to 20-fold less than typically used for transfection of your experimental cell lines. We have found that Silencer Select siRNAs reduced mRNA levels >80% at final concentrations of 2-10 nM using lipid-mediated transfection in HeLa and U2OS human osteosarcoma cells.

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It is expected that 800 plates of 96 wells would be used for a genome-wide screen. Allowing for 3 biological replicates, the total number of wells to be transfected are: 800 plates x 96 wells x 3 replicates = 230,400 wells.
0.15 µL of Lipofectamine RNAiMAX reagent needs to be added per well = 34,560 µL of Lipofectamine RNAiMAX reagent= approximately 35 mL total of Lipofectamine RNAiMAX reagent for a single genome-wide screen with 3 replicates.

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For those who want to re-seal plates after resuspension, we recommend aluminum seals over plastic. We have used the following seal in the past with good success:

- Thermo Scientific Nunc Microplate Lids; Thermo Scientific Cat. No. 250002
- Sealing Films, Axygen Scientific, (Cat. No. 47734-816), Description: Aluminum Sealing Film. Maintains excellent seal on microplates under the widest temperature conditions ( -80° to 97 degrees C) and reagents. Ideal for light-sensitive samples. Uniformly applied adhesive.

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RNA oligonucleotides are susceptible to degradation by exogenous ribonucleases introduced during handling. Wear gloves when handling this product. Use RNase-free reagents, tubes, and barrier pipette tips. Upon receipt, your siRNAs may be safely stored in a non-frost-free freezer at or below -20 degrees C (dried oligonucleotides are shipped at ambient temperature). Invitrogen siRNA reagents are shipped as dry pellets at ambient temperature and should be stored at -20 degrees C upon arrival in a manual defrost or noncycling freezer. Under these conditions, the siRNAs are stable for at least 3 years. If necessary siRNAs as dry pellets (unopened) can be stored at 4 degrees C for at least a year.

To resuspend Invitrogen siRNAs provided in plates:
Centrifuge each plate at low speed (maximum RCF 4,000 x g) to collect the contents at the bottom of the wells before removing the seal.
Wipe the adhesive foil cover with 70% ethanol or other RNase-decontamination solution such as RNaseZap RNase Decontamination Solution (Cat. No. AM9780, AM9782, AM9784).
1. Thermo Scientific or carefully peel back the foil seal to gain access to wells. Use caution and avoid shredding the seal.
2. Add nuclease-free, sterile water, using a multichannel pipettor or liquid handling system and sterile tips, to achieve the desired concentration. Resuspend siRNAs to a convenient stock concentration using the recommended volume of Invitrogen Nuclease-Free Water (not DEPC-treated). Concentrated stocks of 10 µM or more are recommended. However, stock solutions of 2-5 µM may better accommodate dilution schemes for high?throughput transfections and assays conducted on robotic platforms.
3. Gently pipet up and down 5 times to resuspend. Place the solution on an orbital mixer/shaker for 70-90 minutes at room temperature. This additional mixing results in more reliable resuspension.
4. Centrifuge briefly to collect the liquid at the bottom of the wells, if necessary.
5. (Optional) Aliquot the siRNAs into one or more daughter plates, to limit the number of freeze/thaw cycles to which the siRNAs are subjected.
6. Store at -20 degrees C or lower for long-term storage. The siRNAs can be stored at 4 degrees C for short-term use, but care should be taken to seal well to avoid evaporation.

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Store in non-frost-free freezer at -20 degrees C or lower for long-term storage. The siRNAs can be stored at 4 degrees C for short-term use up to a week, but care should be taken to seal well to avoid evaporation. siRNA libraries can be stored at -20 degrees C as resuspended siRNA at less than or equal to 1 µM for one year. Thaw siRNA plates on the benchtop and centrifuge briefly to collect all liquid prior to opening seals.

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Invitrogen siRNA libraries are shipped dried and are therefore shipped at ambient temperature. Although dried siRNAs are remarkably stable, we recommend that you store the dried siRNAs at -20 degrees C (or lower) until ready for use.

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It is recommended that working or screening plates are not subjected multiple freeze/thaw cycles. You can temporarily store well-sealed working plates at 4 degrees C for up to 1 week, spin plates down briefly to collect liquid in the bottom of the tubes. Working and screening siRNA in are typically plated in much lower concentrations than the stock siRNA plates and are more susceptible to degradation under freeze/thaw conditions. For best results, only thaw screening plates when you are ready to add transfection reagents and cells for RNAi screening protocols.

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Once you have assembled all the necessary plasticware and diluents, the siRNA can be distributed into sterile tissue culture plates suitable for RNAi screening protocols. We recommend that you aliquot the siRNA as a droplet onto the working surface of the sterile tissue culture plate, then seal and freeze the plates at -20 degrees C immediately to reduce risk of contamination or excessive evaporation. siRNA that evaporate are more difficult to complex with most transfection reagents which can lead to decreased transfection efficiencies. You can aliquot larger volume with less concentrated working stock if excessive evaporation is observed in your operation. In most cases, up to 20 µL of siRNA for a 96-well plate, or 10 µL of siRNA for a 384-well plate, is still compatible with a wide variety of cells and transfection reagents.

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Library stock plates are usually resuspended at less than or equal to 1 µM and stored at -20 degrees C or lower. Optimal storage is achieved at 10 µM stock concentrations. For Silencer siRNA, we recommend that the number of freeze/thaw cycles of the stock plates is limited to less than 10 for best results. For Silencer Select siRNA, we recommend that the number of freeze/thaw cycles of the stock plates is limited to less than 50 for best results.

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Please see the protocol below to resuspend Silencer and Silencer Select siRNAs (provided as dried powder) in plates:

1. Centrifuge each plate at low speed (maximum RCF 4,000 x g) to collect the contents at the bottom of the wells before removing the seal.
2. Remove seal carefully. To prevent the glue to stick to the plate, you may want to remove the seal while the plate is still cold (from the -20 degrees C or -80 degrees C).
3. Add nuclease-free, sterile water, using a multichannel pipettor or liquid handling system and sterile tips, to achieve the desired concentration (typically 2-10 µM).
4. Gently pipet up and down 5 times to resuspend, and incubate at room temperature for at least 10 minutes to resuspend completely.
5. Centrifuge briefly to collect the liquid at the bottom of the wells, if necessary.
6. Aliquot the siRNAs into one or more siRNA working plates to limit the number of freeze/thaw cycles to which the siRNAs are subjected. Care should be taken to track the number of freeze/thaw cycles.
7. Place a new sterile seal (such as Axygen Cat. No. PCR-AS-200) on the plate before storing.
8. Store in a non-frost-free freezer at -20 degrees C or lower for long-term storage. The siRNAs can be stored at 4 degrees C for short-term use, but care should be taken to seal well to avoid evaporation.


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We recommend resuspending your siRNAs at greater than or equal to 1 µM.

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A stock plate is the original resuspended siRNA, usually ordered for long-term storage of siRNA libraries, in the concentration of <1-10 µM. siRNA working plates are of intermediate concentration of siRNA used to stamp out screening siRNA plates for cell culture and siRNA screening procedures. They are usually in concentrations between 50-200 nM. siRNA screening plates are typically tissue culture plates containing a specified amount of siRNA appropriate for RNAi screening protocols. They are usually in the concentration of 0.5-2.5 pmol of siRNA in a small volume (<20 µL of water). Transfection reagents and cells are directly added to screening plates for reverse transfection procedures.

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We quantify and aliquot our oligos using controlled processes and validated instrumentation. Every plate also undergoes a visual inspection prior to shipment.

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Identity: The mass of a sample of each single-stranded RNA oligonucleotide is analyzed using MALDI-TOF mass spectrometry and compared to the calculated mass.
Annealing: A sample of the annealed siRNA is analyzed by nondenaturing gel electrophoresis.

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Our siRNA libraries are compatible with most automated liquid handling robots including Tecan, Hamilton, and custom-modified robots. Our R&D teams use Tecan liquid handlers with the following plates:

- 96-well plates, Axygen Cat. No. PCR-96-FS-C, 96 Well Full Skirt PCR Microplates
- 384-well plates, Axygen Cat. No. P-384-120SQ-C, 120 µL 384 Deep Well "Diamond Plate" Microplate with Square Wells, Clear.

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There are many acceptable methods for preparing your siRNA plates, and each user may have unique needs. These unique needs should determine how you work up your siRNA library. It is best to have your strategy for library handling worked out, and answers to the following questions in hand prior to resuspending the stock plates:

1. Will you create a backup copy of your library? Keep in mind that this approach will double the freezer storage space and plasticware, tips, and foil seals needed for the backup stock siRNA library, but you will have a backup in case of need.
2. Will you generate working or screening plates at the time of initial resuspension of the siRNA stock plates? If not, tightly seal all stock plates. The stock siRNA libraries can be stored long term in a non-frost-free freezer at -20 degrees C or lower.
3. If you will be generating working or screening plates at the time of resuspension, how many screening plate copies will you generate? Do you have the appropriate freezer space to accommodate these copies?
4. What final concentration of siRNA will you be using in your siRNA screening plates? What transfection reagent will you use? What cell type will you use? In most cases, Silencer siRNA libraries can be screened at 30 nM and Silencer Select siRNA libraries can be screened at 5 nM for most commonly available immortalized adherent cell lines (like HeLa and U2OS cell lines). For best results, the final concentration of your siRNA in your screening plates should be determined empirically through transfection optimization experiments in your cell types of choice using selected siRNAs for this purpose.

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