RNAi Synthetics Support—Getting Started

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.

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.

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

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.

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

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

We recommend the following controls:

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

Please see the following:

(a) Silencer® Select siRNA: “Life Technologies 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: “Life Technologies 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: “Life Technologies 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, Life Technologies 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 Ambion® 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.”

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.

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).

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.

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 β-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 β-galactosidase, enabling you to use β-Gal levels to measure the amount of RNAi-induced degradation of the target gene.

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.

Yes, sets of siRNAs can be ordered in 96-well or 384-well plates. 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. Please email your request to RNAilibraries@lifetech.com.

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.  

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®, Cy®3), custom sizes (>50 nmol) and aliquotting. Custom modified siRNAs are not guaranteed.

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. 

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

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. 

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 β-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. 

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.

Silencer® siRNAs were our first-generation siRNAs, while Silencer® Select siRNAs were our second generation of Ambion® 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.

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.

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.

Yes, siRNA is available for sequences 21 bases in length with or without Silencer® Select modifications.  For longer or shorter siRNA, please email oligos@ambion.com.  

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

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 customSilencer® Select library services and/orSilencer® Libraries for human and mouse.

Yes, our Silencer® Select siRNA are designed to target both cytoplasmic and nuclear long (>100nt) ncRNAs for human, mouse and rat.  ncRNAS for which pre-designed siRNA are available closely match the TaqMan® Non-coding RNA assays currently offered by Thermo Fisher Scientific.

We offer Cy®3 and FAM® labeled Silencer® siRNA for both Predesigned/Validated and Custom Synthesized Silencer® siRNA, once HPLC is chosen as the purification or by requesting a quote. 

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

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

No, Silencer® Select siRNA cannot be ordered with fluorescent labels. While we have not tested it in house, Silencer® Select siRNAs should be labeled efficiently with the Silencer® Labeling Kits (Cat. No. AM1632 and AM1634). 

We offer several negative and positive controls for both Silencer® and Silencer® Select siRNA.

For Silencer® siRNA positive control:

- Silencer® Firefly Luciferase (GL2 + GL3) siRNA (Cat. No. AM429, 5nmol)

- Silencer® GAPDH siRNA (human) (Cat. No AM4605, 5nmol; AM4633, 40 nmol)

- Silencer® GAPDH siRNA (human, mouse, rat) (Cat. No. AM4624, 5 nmol; AM4631, 40 nmol; AM4632, 5 x 40 nmol)

- Silencer® GFP (eGFP) siRNA (Cat. No. AM4626, 5 nmol)

- Silencer® KIF11 (Eg5) siRNA (human, mouse, rat) (Cat. No. AM4639, 5 nmol)

If working with Fluorescently labeled Silencer® siRNA:

- Silencer® Cy™-3 labeled GAPDH siRNA (human, mouse, rat) (Cat. No. AM4649, 5 nmol)

- Silencer® FAM®-labeled GAPDH siRNA (human, mouse, rat) (Cat. NO. AM4650, 5 nmol)

If working with Silencer® siRNA in vivo ready:

- Silencer® GAPDH Positive Control siRNA, in vivo ready (Cat. No. 4404025, 250 nmol)

For Silencer® siRNA negative controls:

- Silencer® Negative Control No. 1 siRNA (Cat. Nos. AM4611, 5 nmol; AM4635, 40 nmol; AM4636, 5 x 40 nmol)

- Silencer® Negative Control No. 2 siRNA (Cat. Nos. AM4613, 5 nmol; AM4637, 30 nmol)

- Silencer® Negative Control No. 3 siRNA (Cat. No. AM4615, 5 nmol)

- Silencer® Negative Control No. 4 siRNA (Cat. No. AM4641, 5 nmol)

- Silencer® Negative Control No. 5 siRNA (Cat. No. AM4642, 5 nmol)

If working with Fluorescently labeled Silencer® siRNA:

- Silencer® Cy™ 3-labeled Negative Control No. 1 siRNA (Cat. No AM4621, 5 nmol)

- Silencer® FAM®-labeled Negative Control No. 1 siRNA (Cat. No. AM4620, 5 nmol)

If working with Silencer® siRNA in vivo ready:

- Silencer® in vivo ready (Cat. No. 4404021)

For Silencer® Select siRNA Positive control:

-  Silencer® Select GAPDH Positive Control siRNA (Cat. No. 4390849, 5 nmol; 4390850, 40 nmol; 4404024, 250 nmol)

-  Silencer® Select MALAT1 Positive Control siRNA for non-coding siRNA (Cat. No. 4455877, 5 nmol)

If working with Silencer® Select siRNA in vivo ready:

- Silencer® Select GAPDH in vivo (Cat. No. 44404024)

For Silencer® Select siRNA negative control:

-  Silencer® Select Negative Control No. 1 siRNA (Cat. No. 4390843, 5 nmol; 4390844, 40 nmol)

-  Silencer® Select Negative Control No. 2 siRNA (Cat. No. 4390846, 5 nmol; 4390847, 40 nmol)

If working with Silencer® Select siRNA in vivo ready:

-  Silencer® Select Negative Control No. 1 siRNA, in vivo ready (Cat. No. 4404020, 250nmol)

A positive control siRNA such as the Silencer® Select GAPDH siRNA will demonstrate efficient transfection and help to optimize transfection conditions. A negative control siRNA is necessary to serve as a baseline for gene expression. Read more about controls here.

We do have a positive control siRNA targeting MALAT-1, which is a ncRNA localized mostly in the nucleus (Cat. No. 4455877).  Silencer® Select Negative Controls #1 and 2 can be used as the negative controls.

Only one Negative Control siRNA is needed. We offer two different negative control sequences to account for the rare case of an off target effect with one sequence.

Stealth RNAi™ is a 25-mer blunt dsRNA oligo that contains a chemically modified sense strand.  While this modification is proprietary, it helps to:

-         Increase stability in the RISC complex

-        Increase stability in vitro

-        Decrease off-targeting effects of sense strand since it won’t be used as the guiding strand

Stealth RNAi™ can be ordered via web or email, and are available at 20 nmol, 80 nmol, 1 µmol, and 1.7 µmol.  The RNAi is delivered lyophilized in annealing buffer, along with 1 mL DEPC water for dilution.

Stealth Select RNAi™ are pre-designed Stealth RNAi™ molecules for mouse, rat or human.  These sequences have been run through Smith-Waterman algorithm to further minimize off target effects.  These sequences are mainly ORF targeted, but have not been bench-tested.  This RNAi can be ordered online, and found on the RNAi Express Search engine.  The top 3 non-overlapping targets per gene are provided, so that a set of three can be purchased.  Sequences are provided on the CoA with delivered product.  20nmol of the annealed duplex will be delivered lyophilized, along with 1 mL DEPC water for dilution.  These RNAi can also be ordered at 1 nmol through plate select.  We guarantee 2 out of 3 Stealth Select RNAi™ siRNA targeted to the same gene will give you 70% or greater knockdown.

Validated Stealth RNAi™ are predesigned and bench-tested Stealth RNAi™.  These RNAi are non-overlapping sequences targeting human genes.   These RNAi are delivered at 20 nmol of each annealed duplex lyophilized, with 1 mL DEPC treated water.  They can also be ordered at 1 nmol through plate select. These siRNA are guaranteed individually.

If a pre-designed siRNA is unavailable, you can use our algorithm to design one using your target mRNA nucleotide sequence or accession number (species other than human, mouse, rate; 5’ or 3’ UTR).  The RNAi Designer is a free online tool that is ideal for designing effective Stealth RNAi™. The RNAi Designer uses a proprietary algorithm based on published design rules, sophisticated homology elimination, and mining of sequence information from Invitrogen’s extensive wet lab data on successful RNAi. Using RNAi Designer, you’ll be able to design a unique Stealth RNAi™ molecule to target your mRNA of interest for effective knockdown. If you have an existing siRNA sequence that works well for you, it is simple to change this to an effective Stealth™ duplex and get all the Stealth RNAi™ benefits. 

The RNAi Designer utilizes stringent design rules to select Stealth™ siRNA that are unique to the organism that you select. Stealth RNAi™ is most effective at minimizing this concern, as only the antisense strand is capable of eliciting the RNAi response.

• Predesigned and Validated
• Custom Designed
• Desalted Custom Synthesized
• HPLC and in vivo purity custom Synthesized

All Stealth siRNA™ duplexes are delivered annealed and lyophilized. 

The Stealth siRNA™ is delivered as 20 nmol of annealed duplex. It offers approximately 4000 transfections of 24-well size dishes (5 pmol/well). Invitrogen™ can synthesize selected sequences in larger scale upon request. 

Stealth Select sets are delivered as 3 separate vials containing each unique duplex.  They are ready to transfect immediately once the lyophilized RNA is rehydrated at the desired concentration.  We recommend that each sequence be transfected and tested individually, as pooling of multiple RNAi duplexes together increases the risk of negative off target effects in the cells, and further analysis is then needed to eliminate which sequences are contributing to the knockdown. 

We offer several negative and positive controls for Stealth siRNA:

For Stealth Positive Control:

-  Stealth RNAi™ siRNA Actin Positive Control (human) (Cat. No. 12935141, 250 µL)

-  Stealth RNAi™ siRNA Cyclophilin B Control (human) (Cat. No. 12935142, 250 µL)

-  Stealth RNAi™ siRNA GAPDH Positive Control (human) (Cat. No. 12935140, 250 µL)

-  Stealth RNAi™ siRNA β-Lactamase Reporter Control (Cat. No. 12935148, 250 µL)

-  Stealth RNAi™ siRNA GFP Reporter Control (Cat. No. 12935145, 250 µL)

-  Stealth RNAi™ siRNA Luciferase Reporter Control (Cat. No. 12935146, 250 µL)

For Stealth Negative Control:

Hi (55-65% GC), Med (45-55% GC), or Low (35-45% GC) Duplexes are offered:

-  Stealth RNAi™ siRNA Negative Control Hi GC (Cat. No. 12935400, 250 µL)

-  Stealth RNAi™ siRNA Negative Control Hi GC Duplex #2 (Cat. No. 12935114, 250 µL)

-  Stealth RNAi™ siRNA Negative Control Hi GC Duplex #3 (Cat. No. 12935115, 250 µL)

-  Stealth RNAi™ siRNA Negative Control Med GC Duplex (Cat. No. 12935300, 250 µL)

-  Stealth RNAi™ siRNA Negative Control Med GC Duplex #2 (Cat. No. 12935112, 250 µL)

-  Stealth RNAi™ siRNA Negative Control Hi GC Duplex #3 (Cat. No. 12935113, 250 µL)

-  Stealth RNAi™ siRNA Negative Control Low GC Duplex (Cat. No. 12935200, 250 µL)

-  Stealth RNAi™ siRNA Negative Control Low GC Duplex #2 (Cat. No. 12935110, 250 µL)

-  Stealth RNAi™ siRNA Negative Control Low GC Duplex #3 (Cat. No. 12935111, 250 µL)

-  Stealth RNAi™ siRNA Negative Control Kit (Cat. No. 12935100, contains one of each GC content level negative control, 250 µL each at 20 µM)

-  For a scrambled negative control, you could also scramble your designed siRNA or Stealth RNAi™ sequence using the BLOCK-iT RNAi Designer.

You can also use our BLOCK-iT AlexaFluor Red Fluorescent Control (Cat. No. 14750100) or BLOCK-iT Fluorescent Oligo (Cat. No. 2013, 13750062) as a control for the transfection.

The easiest way to do this is to use the identification numbers (HSS, RSS, MSS) and go to “Stealth Quick Order” link online.  Sequences that were provided after purchase can also be re-ordered as “custom” stealth molecules online. 

The average molecular weight is 16,100 g/mol.  

If in vivo purity and/or 5’ sense label (Biotin, Alexa Fluor 488, 546, 555, 647, 680, 750) is required, you can order the siRNA through our RNAi in vivo Designer page.   These siRNA can be ordered as HLPC or desalted in vivo purity or HPLC in vivo purity.

You could order a Block-it RNA oligo or custom RNA oligo.  Block-it RNA oligos are small 21-23 unmodified, nucleotide sequence-specific RNA duplexes.  To design and order BLOCK-iT™ siRNAs, please visit the BLOCK-iT™ RNAi Designer.  Custom siRNAs can also be ordered by email.

Custom RNA oligos can be ordered as single stranded oligos or duplexed to create a double stranded RNA.  The oligo can be up to 50 bp in length, with the option of a biotin, fluorescein or phosphate 5’ modification.  These oligos will be delivered at desalted purity only.  To order custom RNA oligos, visit this website.

Please provide either the accession number or nucleotide sequence.  You will also have the choice of the region of the gene you would like to target:  ORF, 5’ UTR, or 3’ UTR and the species.  The percentage G/C content of the siRNA can also be chosen.  Lastly, you can have your input sequence blasted against sequences in the database to ensure unique regions are used to design the siRNAs.

Please see the different siRNA we offer for in vivo studies:

Ambion® In Vivo Pre- designed siRNAs are designed using the Silencer® Select algorithm and incorporate additional chemical modifications for superior serum stability (half life >5 hours at 37 °C in 90% mouse serum) with in vivo applications. Ambion® In Vivo siRNAs are non-toxic and non-immunogenic in vitro (peripheral blood nononuclear cells; PBMC) and in vivo (mouse). In cell-based assays, Ambion® In Vivo siRNAs exhibit potency and specificity equivalent to Silencer® Select siRNAs.

Ambion® In Vivo Custom Designed siRNAs are designed to your specified target, using the Silencer® Select algorithm and Ambion® In Vivo chemical modifications.

Ambion® In Vivo Custom Synthesized siRNAs are synthesized with your sequence, with Ambion® In Vivo chemical modifications for serum stability.

Our Ambion® in vivo siRNA can be ordered with the following purities:

Desalted: Desalted and analyzed by MALDI-TOF mass spectrometry.

HPLC: HPLC purified and analyzed by MALDI-TOF and analytical HPLC

In Vivo Ready : HPLC purified, analyzed by MALDI-TOF and analytical HPLC, dialyzed to remove salts, sterile filtered, and endotoxin tested

Use our GeneAssist tool to order your custom siRNA.

• >100x more stable than unmodified siRNA — longer knockdown in vivo
• Compatible with any 21-mer sequence — no loss of potency
• Non-toxic — actually reduces siRNA sequence specific immune responses
• Enhanced specificity — up to 90% reduction in off target effects
• Both Pre-designed and Validated Ambion® In Vivo siRNAs offered

 Chemically modified siRNA duplexes have a number of advantages over standard siRNA duplexes, including the minimization of off-target effects, enhanced stability, and reduced toxicity. For these reasons, chemically modified siRNA duplexes are recommended for in vivo siRNA experiments. For some pilot experiments, where the only aim is to determine biodistribution, unmodified siRNA and fluorescent controls are useful and slightly more cost-effective. 

We have demonstrated that labeling Stealth  and Silencer® siRNA does not hamper their knockdown potency. An alternative approach is to mix unlabeled duplexes with labeled control duplexes; this method is more commonly used with in vivo siRNA, and allows progression to clinical research unhindered by questions about the possible effects of a label. 

For Stealth siRNA we recommend to select HPLC in vivo purity. For Ambion® In Vivo siRNA, we recommend  in vivo ready purity.

RNAi can be delivered using two different approaches-siRNA synthetic duplexes or siRNA expressed from plasmids or viral vectors (shRNA, miRNAi). siRNA are becoming the method of choice for the fast development of therapeutics. They are easy to use, easy to design, and easy to synthesize. siRNA can be rapidly identified and multiple genes can be targeted at the same time. With RNAi vectors, the expression will be steadier as a result of the possibility of stable integration of the plasmid into the genome, and they have the ability to target nondividing cells such as stem cells, lymphocytes and neurons. The drawbacks are the danger of oncogenic transformation from insertional mutagenesis, and unanticipated toxicity from long-term silencing of human genes and/or having high amounts of siRNA inside the cell (Grimm D. et al.: Nature 441: 537-541 (2006)). 

Several different approaches have been used for siRNA delivery, including various local delivery techniques and systemic delivery. We offer a lipid reagent, Invivofectamine® 2.0 reagent for in vivo siRNA systemic delivery.  Learn more about this reagent here.

 Please contact rnairesearcher@invitrogen.com regarding large scale in vivo siRNA or fill out the quote request form. 

It is recommended to reduce the chance of eliciting an immune response.

Stealth siRNA duplexes for in vivo experiments can be ordered using the BLOCK-iT™ RNAi Express for in vivo synthetics, while Ambion® in vivo siRNA can be ordered using this tool.

Stealth siRNA is markedly more stable in serum than unmodified siRNA. This increased stability is particularly beneficial in vivo where the RNAi may be exposed to more proteases. In collaboration with Intradigm, Stealth RNAi™ has been demonstrated to have activity in vivo when injected intra-tumorally. 

We offer the following in vivo siRNA controls:

• Ambion® In Vivo Negative Control #1 siRNA (Cat. No. 4457287, 5nmol; 4459405, 50 nmol; 4457289, 250 nmol)
• Ambion® In Vivo GAPDH Positive control siRNA (Cat. No. 4457288, 5 nmol; 4459407, 50 nmol, 4457291, 250 nmol)
• Ambion® In Vivo Factor VII Positive control siRNA (Cat. No. 4459408, 50 nmol; 4457292, 250 nmol)
• Silencer® Select Negative Control No. 1 siRNA, in vivo ready (Cat. No. 4404020, 250 nmol)
• Silencer® GAPDH Positive Control siRNA, in vivo ready (Cat. No. 4404025, 250 nmol)

miRNA (microRNAs) are short, highly conserved small noncoding RNA molecules naturally occurring in the genomes of plants and animals.  miRNAs are single stranded,  18-25 nucleotides long and regulate posttranscriptional mRNA expression, typically by binding to the 3’ untranslated region (3’-UTR) of the complementary mRNA sequence, resulting in translational repression and gene silencing.  Studies have shown that thousands of human protein-coding genes are regulated by miRNAs, indicating that miRNAs are “master regulators” of many important biological processes.

siRNA mediates degradation of a target mRNA molecule in a sequence-specific manner, while miRNA suppresses translation without cleaving mRNA.

Ambion® Anti-miR inhibitors & Ambion® Pre-miR miRNA Precursors are our first generation mimic and inhibitors.  Ambion® Anti-miR™ miRNA Inhibitors are chemically modified, single stranded nucleic acids designed to specifically bind and inhibit endogenous microRNA (miRNA) molecules.  These are ready to use inhibitors that can be introduced into cells using transfection or electroporation.  Pre-designed inhibitors have been created for all miRNAs listed in miRBase version 15.0. 

Ambion® Pre-miR miRNA Precursors are small, chemically modified double-stranded RNA molecules designed to mimic endogenous mature miRNAs. These ready-to-use miRNA mimics, which are similar to, but not identical to siRNAs, can be introduced into cells using transfection or electroporation parameters similar to those used for siRNAs.  Please note, Pre-miR miRNA Precursors are not hairpin constructs and should not be confused with pre-miRNAs.

mirVana™ miRNA mimics and inhibitors are our second generation of mimics and inhibitors.  mirVana™ miRNA mimics are small, chemically double-stranded RNAs that mimic endogenous mature miRNAs and enable miRNA functional analysis by up-regulation of miRNA activity.  They have enhanced specificity over the first generation mimics, are delivered as HPLC purified, and ready to transfect.

miRNA inhibitors are small, chemically modified single-stranded RNA molecules designed to specifically bind to and inhibit endogenous miRNA molecules and enable miRNA functional analysis by down-regulation of miRNA activity.  They have enhanced potency over our first generation inhibitors.  Pre-designed inhibitors were created for all miRNAs listed in miRBase version 19.0.  They come delivered as HPLC purified, and ready to transfect.  Learn more about mirVana miRNA mimics and inhibitors through this video.  Both mimics and inhibitors are available individually or as libraries. 

They are compatible with both in vitro and in vivo applications, and have been validated with Lipofectamine® RNAiMAX Transfection Reagent for use in cell-based systems, and with Invivofectamine® 2.0 Reagent for in vivo delivery. These oligonucleotides are nontoxic and do not induce an immune response in the animal models tested. In vivo–ready mirVana™ miRNA mimics and inhibitors have been purified by HPLC and dialysis and are ready for immediate in vivo use.

Click on the following links to order Ambion® Pre-miR miRNA Precursors, Ambion® Anti-miR miRNA inhibitors, or mirVana™ Mimics & Inhibitors.

Click here to see a table comparing these 4 products. For all library inquiries, please email: rnailibraries@lifetech.com.

Yes, submit your unpublished miRNA sequences and/or order custom modified miRNA here.

We can label our Pre-miR and Anti-miR miRNA, but do not offer labeled mirVana™ Mimics and inhibitors.  5’ modifications include Alexa Fluor 555, Alexa Fluor 647, FAM®, and Cy®3.  Request a quote here.

Please visit miRBase: The microRNA database.

The average molecular weight for mirVana miRNA mimics is 14,000 g/mol (where 1 nmol = 14 µg), and 11,000 – 12,000 g/mol (where 1 nmol = 11 – 12 µg) for mirVana miRNA inhibitors.  The exact molecular weights are proprietary.

Yes, we offer both positive and negative controls for our mirVana™ miRNA mimics.  The mirVana™ miRNA Mimic miR-1 Positive Control (Cat. No. 4464062, 4462063, 4462064, 2262065) is designed to mimic mature miR-1 miRNA after delivery to mammalian cells. miR-1 is associated with down-regulation of many genes. In particular, miR-1-mediated down-regulation of Protein Tyrosine Kinase 9 (PTK9) has been shown to occur at the mRNA level. miR-1 Positive Control is intended for use as a positive control for transfection experiments using mirVana™ miRNA Mimics. Monitor activity of miR-1 Positive Control through its gene silencing of PTK9, using real-time reverse transcription-PCR to detect PTK9 mRNA.

miR-1 miRNA PTK9 Target Information

*For real-time RT-PCR to detect PTK9 mRNA

mirVana™ miRNA Mimic Negative Control #1 (4464058, 4464059, 4464060, 4464061) is designed to use as a negative control for experiments using mirVana™ miRNA Mimics. Transfect the Negative Control using the same methodology as for your positive control (such as miR-1 Positive Control) and experimental mirVana™ miRNA Mimics. Use target gene expression from Negative Control-transfected samples as a baseline for evaluation of the effect of the control and experimental miRNA mimic on target gene expression. mirVana™ miRNA Mimic Negative Control #1 is a random sequence miRNA mimic molecule that has been extensively tested in human cell lines and tissues and validated to not produce identifiable effects on known miRNA function.

Yes, we offer both positive and negative controls for our mirVana™ miRNA inhibitors. The mirVana™ miRNA Inhibitor let-7c Positive Control is intended for use as a positive control for mammalian cell culture experiments using mirVana™ miRNA Inhibitors (Cat. No. 4464080, 4464081).  Endogenous let-7c miRNA has been shown to negatively regulate HMGA2 mRNA in cultured cells. HMGA2 is a ubiquitously expressed, non-histone, chromatin protein that can modulate gene expression through changes in chromatin architecture. let-7c miRNA down-regulates levels of HMGA2 mRNA. When transfected into human and mouse cell lines, let-7c miRNA inhibitor blocks endogenous let-7c miRNA, resulting in increased levels of HMGA2 mRNA. Thus, let-7c miRNA inhibitor activity can be monitored in human or mouse cells using real-time reverse transcription PCR (RT-PCR) to detect HMGA2 mRNA.

mirVana™ miRNA Inhibitor Negative Control #1 is intended for use as a negative control for experiments using mirVana™ miRNA Inhibitors (Cat. No. 4464076, 4464077, 4464078, 4464079).  Transfect this negative control in parallel with your positive control (such as let-7c Positive Control) and experimental mirVana™ miRNA Inhibitors. Use target expression from negative control-transfected samples as a baseline for evaluation of the effect of the control and experimental miRNA inhibitor on target gene expression.

Store the dried oligonucleotides in a non-frost-free freezer at or below –20°C. Store resuspended oligonucleotides at or below –20°C for extended periods (up to 1 year), and they can undergo up to 50 freeze/thaw cycles without significant degradation. We recommend aliquoting the resuspended miRNA to prevent contamination. Storage at –80°C is fine but not necessary. Working stock solutions can be stored at 4°C for up to 7 days before use.

Please email techsupport@lifetech.com with your request. Negative controls require proof of purchase prior to release.

Yes, please provide us with your unpublished miRNA sequence and we can synthesize a corresponding mirVana™ mimic or inhibitor by clicking on this link.

The turnaround time varies depending on the level of purification and whether it is a custom order or not. Please see the approximate timetable below:

Mimics and inhibitors can be transfected or electroporated into cells using parameters similar to those used for siRNA.

Using mirVana™ miRNA Mimics

For the following applications, use a cell line that expresses relatively low levels of the mature miRNA of interest.

• Cell density at the time of transfection. In general, 30–70% confluency is recommended.

• Transfection method (traditional or reverse transfection).

• Length of exposure of cells to transfection agent-RNA oligonucleotides complexes.

For many miRNA mimics, maximal activity is achieved after 24 hours, but biological assays may be performed 24–72 hours posttransfection. If you maintain your transfected cells longer than 24 hours, we suggest replacing the existing medium with fresh medium at 24 hours posttransfection, for greater viability of the cells.

Using mirVana™ miRNA Inhibitors to analyze endogenous miRNA function

For the following applications, use a cell line that expresses relatively high levels of the mature miRNA of interest.

General transfection starting points

mirVana™ miRNA Mimics and Inhibitors typically work best when transfected at a final concentration of 3–30 nM. Optimization experiments might include a concentration range of 1–100 nM. To increase accuracy and reproducibility when preparing transfection complexes, prepare a dilution of your stock oligonucleotides, and pipet a higher volume of diluted stock.

Using TaqMan® miRNA assays, you can measure the level of miRNA of interest. If the miRNA is bound to the synthetic anti-miRs and/or mirVana™ inhibitors transfected, the duplex should not be detected, indicating efficient miRNA inactivation. Please note, this only works when total RNA sample isolated is not heat denatured when RT primer is added, as heat denaturation will result in complex dissociation and subsequent detection of microRNA by TaqMan® miRNA assay even when cells have been transfected properly. Please see the protocol for the TaqMan® MicroRNA Cells-to-C_T kit (Cat. No. 4391848) for more details.

Please see the controls we offer below for in vivo miRNA studies:

• mirVana™ miRNA mimic, mir-1 positive control (25 nmol, 4464065)
• mirVana™ miRNA mimic negative control Number 1 (250 nmol, 4464061)
• mirVana™ inhibitor negative control Number 1 (250 nmol, 4464079)

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

The chemistry of RNA synthesis is identical to the DNA synthesis except for the presence of an additional protecting group at the 2' hydroxyl position of ribose. This position is protected with silyl groups, which are stable throughout the synthesis. The remaining positions on both the sugar and the bases are protected in the same fashion as in DNA. 

The RNA can be purified by analytical HPLC. Alternatively the RNA can be ordered desalted, which is the default purity. We do not offer additional purification post-annealing of duplexes. 

Desalting means the salts of synthesis are removed but not failure sequences. HPLC purification means the full-length oligo is purified and the shorter failure sequences are removed.

The current maximum length of RNA oligonucleotides is 50 bases. 

98.5% 

The offered synthesis scales are 50 nM and 200 nM. The synthesis scales refer to the amount of starting material present, not the amount of final product produced.  Please view our minimum yield guarantee for the amount of RNA oligo we guarantee based on scale and purification.

RNA oligos are delivered lyophilized. 

For single-stranded RNA, the product documentation includes the mass, moles, and OD numbers for each RNA oligo. For duplex RNA, the amount of product delivered is listed on the tube.

Please reference the 5’ and 3’ modification options on the RNA oligo ordering page.

Yes. Simply include TT in the requested sequence. 

The best way to store RNA is as a dry pellet at –20°C or –80°C. 

As a dry pellet they can be stored at –20°C for 6 months. 

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.