RNAi Libraries Support—Getting Started

Yes, sets of siRNAs can be ordered in a 96well or 384-well plate. siRNA libraries can target any number of genes, e.g., an entire genome, a gene family, a biological pathway, or a custom set of genes.

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°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.

Our R&D teams use:

• 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.

http://www.axygen.com/core_products/384-square-deep-well-plate-120ul

• 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.

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

Please visit our Transfection Support Center to find technical resources, tips and tricks, and troubleshooting information on transfection.

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.

We recommend resuspending your siRNAs at ≥1 μM.

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°C or –80°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). (See table below.)

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°C or lower for long-term storage. The siRNAs can be stored at 4°C for short-term use, but care should be taken to seal well to avoid evaporation.

We recommend resuspending your siRNAs at ≥1 μM; 10 μM is best for long-term storage. Use the table below for guidelines for resuspending your siRNA libraries.

*** Volume exceeds the capacity of the tube

Library stock plates are usually resuspended at ≤1 µM and stored at –20°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.

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°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.

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°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.

Ambion 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°C (or lower) until ready for use.

Store in non–frost-free freezer at –20°C or lower for long-term storage. The siRNAs can be stored at 4°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°C as resuspended siRNA at ≤1 μM for one year. Thaw siRNA plates on the benchtop and centrifuge briefly to collect all liquid prior to opening seals.

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°C (dried oligonucleotides are shipped at ambient temperature).

Ambion siRNA reagents are shipped as dry pellets at ambient temperature and should be stored at –20°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°C for at least a year.

To resuspend Ambion 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. Pierce 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 Ambion 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°C or lower for long-term storage. The siRNAs can be stored at 4°C for short-term use, but care should be taken to seal well to avoid evaporation.

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°C) and reagents. Ideal for light-sensitive samples. Uniformly applied adhesive.

The efficiency with which mammalian cells are transfected with siRNA will vary according to cell type and the transfection agent used. The major variables that impact siRNA transfection efficiency are the following: transfection reagent type and amount, # of cells plated in well, type of siRNA, concentration of siRNA. The chart below gives a sample of transfection variable for typical transfection conditions for HeLa cells is given. Optimal conditions for the cell types of interest for your siRNA screening should be determined empirically using pilot siRNA screens. Since Silencer Select siRNAs exhibit superior silencing potency compared to other siRNA libraries, we suggest Silencer Select siRNA concentrations of 5- to 10-fold less than typically used for other siRNA technologies. 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.

For Silencer Select siRNA, a typical transfection setup may be the following:

For Silencer siRNA (unmodified siRNA), a typical transfection set up may be the following:

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= ~35 mL total of Lipofectamine RNAiMAX reagent for a single genome-wide screen with 3 replicates.

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.

^a Lipofectamine RNAiMAX Transfection Reagent recommended. Refer to the instructions provided with your transfection agent for the recommended volume. ^bThe amounts indicated result in a final oligonucleotide concentration of 30 nM.

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 predetermined 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 nontargeting negative control siRNA (e.g., Silencer Select Negative Control #1)—data from these crucial controls serve as a baseline for evaluation of experimental target knockdown.

Untransfected 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 nontargeting negative control siRNA, valuable information about the effects of transfection on cell viability can be obtained.

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

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.

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.

We generally don't recommend pooling. However, it is up to the customer to design the experiments that fits best for their needs and limitations. 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 been targeting 3‘ UTR and acting like miRNA, it is not an issue with newer generation 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.

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.

If you have already identified a desired transfection reagent but still want to fine-tune transfection conditions or monitor transfection efficiency in your experiment, consider using Silencer Select positive and negative control siRNAs.

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

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 >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.

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 RNAiSupport@thermofisher.com.

Below is a pictorial depiction of the different library subsets and their organization into larger sets for the Silencer Select library:

These sets are usually in stock and available for immediate shipment. See below for formatting details.

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.

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.

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 Ambion 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.

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.

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.

It‘s probably easiest to see an example. In the Silencer Human Phosphatase siRNA Library V3, there are 12 plates of siRNAs:

As shown below the plates designated ‗A1-1‘ has siRNAs (think of them as ―A siRNA designs‖ to the exact same targets as the plates designated ‗B1-1‘ (the ―B siRNA designs‖) and ‗C1-1‘ (the ―C siRNA designs‖ and those siRNAs are plated in the same target order. Likewise, plates ‗A1-2‘, ‗B1-2‘, and ‗C1-2‘ all have siRNAs to the same exact targets.

If you wanted to run a screen with pools of siRNAs, you would pool the each of the same well locations of the A1-1, B1-1, and C1-1 plates.

Note that Plate IDs are different from Plate Names. Each Plate ID is a unique 8-digit code with ―CPF. The Plate ID is what is printed on the barcode in ―3 of 9‖ format. Both Plate Name and Plate ID information is provided in the file shipped with your siRNA Library.

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‘ll 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).

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 Life Technologies Technical Support at RNAiSupport@thermofisher.com and provide the Lot Number of your siRNA library.

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.

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

We use a heat-seal system to affix the seals on Ambion 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°C or –80°C freezer).

Below are some citations that reference Ambion siRNA libraries/collections. This is only a subset. Please contact us for an updated citation list at RNAiSupport@thermofisher.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 β-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.

Contact your local sales representative or contact us at RNAiSupport@thermofisher.com. We‘ll provide you with a Quote Request form to indicate exactly how you would like your siRNA library formatted. You‘ll 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 Ambion 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

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.

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.

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.

Turnaround times vary depending on the number of siRNAs and the complexity of the plating request. Most Ambion 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 RNAiSupport@thermofisher.com for more information or a quote.

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.

The figure above displays: Silencer Select siRNAs elicit expected phenotype at a higher rate than other siRNAs. siRNAs to seven gene targets with well-understood RNAi induced phenotypes were individually transfected at 3 nM, and the phenotypes were measured 48 hours later. Each bar represents the percent of siRNAs that gave the expected, silenced phenotype. siRNAs to BUB1B, AURKB, WEE1, and PLK1 were assessed using a multi-parametric cell growth/apoptosis assay in U2OS human osteosarcoma cells. siRNAs to HMGCR, LDLR, and FDFT1 were assessed using an LDL uptake assay in HUH7 human hepatoma cells.

You therefore need fewer individual siRNAs in your screen to get the confirmatory results that you need to have confidence in moving forward with your hits. By using 3 highly effective siRNAs rather than 4 less effective siRNAs, you can cut your screening costs by up to a third.

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.