RNAi FAQs

Questions about Stealth RNAi:

What is Stealth RNAi?
Stealth RNAi is the next generation in RNAi chemistry. Stealth RNAi molecules are 25mer, blunt-ended duplexes with proprietary chemical modifications. These chemical modifications were specifically designed to eliminate the induction of cellular stress response. They also make Stealth RNAi more stable than standard siRNA in serum and reduce the possibility of off-target effects by ensuring that only the antisense strand of the Stealth duplex enters the RNAi pathway.

How do I design Stealth RNAi?
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.

How do I know the siRNA or Stealth RNAi will not target other genes?
The RNAi Designer utilizes stringent design rules to select Stealth RNAi that are unique to the organism that you select. Stealth RNAi is most effective at eliminating this concern, as only the antisense strand is capable of eliciting the RNAi response.

How do I order Stealth RNAi?
The best way to order Stealth RNAi is to use the RNAi Designer on Invitrogen’s website. The selected 25 bp Stealth RNAi duplexes are delivered annealed and lyophilized.

How much Stealth RNAi do I need?
The Stealth RNAi is delivered as 5 nmol of annealed duplex. It offers approximately 500 transfections of 24-well size dishes. Invitrogen can synthesize selected sequences in larger scale upon request.

How do I know what sequence to use?
You may use the RNAi Designer online tool to identify Stealth RNAi sequences. Invitrogen Custom Services can also help you design and validate sequences with cell types of interest.

How stable is Stealth RNAi, can I use it for in vivo research?
Stealth RNAi is markedly more stable in serum than 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 intratumorally.

Is the fluorescent oligo in the BLOCK-iT Basic RNAi Control Kit and Transfection Optimization Kit modified as Stealth RNAi?
The fluorescent oligo is modified fluorescein-labeled dsRNA. The modifications on this RNA make it a great control for transfection—causing it to localize to the nucleus in a clear and stable manner. In contrast, simply labeling siRNA with Fluorescein, results in an ill-defined punctuate pattern that which makes it difficult to distinguish whether fluorescent oligos are internalized or simply sticking to the outside of the cells.

Could I use the BLOCK-iT Fluorescent Oligo to optimize plasmid transfection?
BLOCK-iT Fluorescent Oligo transfection has been shown to correlate with Stealth RNAi and siRNA transfection. However, no correlation between DNA and the BLOCK-iT Fluorescent Oligo has been tested.

General RNAi Questions:

How was RNA interference (RNAi) discovered?
The first indication in 1990 of the existence of the RNAi phenomenon was in work completed by Jorgenson and Mol in a plant model system. Petunias that had an additional copy of a native pigment gene somehow seemed to block the expression of both the native and the inserted pigment gene, called co-suppression. A few years later, working in tobacco, another group found that viruses could trigger the suppression of specific genes. The term RNAi interference was coined in a 1998 Fire et. al Nature paper, after the discovery in the labs of Andrew Z. Fire and Craig C. Mello that injecting dsRNA into C. elegans led to the silencing of expression of those specific genes homologous to the dsRNA delivered.

What is the natural biological function of RNAi?
It seems that nearly all plant and animal cells have the internal machinery to use siRNA in a function to suppress the expression of particular genes. The RNAi machinery appears to have evolved to protect the genome from endogenous transposable elements and from viral infections. RNAi may also have a normal functional role in cells to inhibit the expression of some genes during growth and development.

What are the advantages of RNAi over other methods used for knocking down gene expression?
Knockout and transgenic animals are key ways that the loss-of-function phenotype has been studied. However, it is a long and expensive process to generate these animals. Also, loss of many genes results in an embryonic lethal phenotype making knockouts impossible.

Methods for inactivating protein function include the construction and analysis of dominant-negative mutants, and the use of antibodies and aptamers. However, these may take some time to identify what form or construct will function, and they appear to work for only certain targets.

Two methods have been used for modulating mRNA turnover, antisense and ribozymes. Both of these methods seem to be limited in their applications.

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.

What are the ways that short interfering RNA (siRNA) can be generated?
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

What is the difference between siRNA and diced siRNA (d-siRNA)?
The structure of the molecules is the same dicer specifically cleaves long dsRNA into the 21-23 nucleotide duplexes with a two-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.

How do I measure an RNAi effect?
The most common way to measure gene specific knockdown is to perform western blot analysis to compare the level of protein expression before and after the introduction of siRNA. In some cases a reporter system that allows easy measurement of a reporter gene, such as b-galactosidase, may be used. Real-time PCR (such as by using LUX Primers ) to measure the level of transcript present in the cell or other types of cell-based assays may be also be employed.

How can I know what sequence is responsible for an RNAi effect?
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 siRNA (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.

I only need to knock down gene expression for <7 days
First try using Stealth RNAi to transfect your cells with Lipofectamine RNAiMAX Transfection Reagent

I cannot get knockdown in my cells with Stealth RNAi and Lipofectamine RNAiMAX
Your cells may not be transfectable with a lipid. Try using miR RNAi vectors with Adenoviral or Lentiviral delivery.

I need to knock down gene expression for >10 days
First try using the BLOCK-iT Pol II miR RNAi Vectors with Lipofectamine 2000 Transfection Reagent

I need to be able to turn the RNAi expression on and off
Use RNAi vectors with the inducible H1/TO promoter.

Questions about BLOCK-iT Vectors:

Why use vectors for shRNA delivery?
In mammalian cells, RNAi can be induced by the direct introduction of molecules that specifically knock down the expression of a selected gene and result in a loss-of-function phenotype. These knockdown molecules can be chemically synthesized, prepared by in vitro methodologies, or produced in a cell from a DNA vector template. The first two of these methods can only be used for transient knockdown and can be difficult to introduce into hard-to-transfect, non-dividing or primary cell types. The use of vectors to deliver a short-hairpin RNA (shRNA) expressed from a pol III promoter has expanded RNAi experimental options to include stable and inducible expression as well as viral delivery.

Why use a pol III type promoter?
For efficient shRNA expression a pol III type promoter is used. These Pol III promoters contain all of their essential elements upstream of the expressed RNA and terminate with a short polythymidine tract. Once the shRNA is expressed, it is transported from the nucleus and processed into siRNA in the cytoplasm by the enzyme Dicer. Dicer preferentially recognizes shRNA generated from a pol III promoter because they carry no 5’ or 3’ flanking sequences. The siRNA enters into RISC complexes and generates an RNAi response in mammalian cells.

What is the difference between the H1 and the U6 promoters?
The BLOCK-iT Inducible H1 and U6 Entry Vector Kits use either the Pol III dependent H1 or the U6 promoter, respectively. The H1 promoter is modified to contain two flanking tetracycline operator 2 (TetO2) sites within the H1 promoter. This allows the shRNA expressed from this promoter to be regulated in cells that express the tetracycline repressor (TR) protein. Both the H1 and the U6 are pol III type promoters; however, there may be some minor differences in their effectiveness depending on the cell line used.

When should the pLenti6/TR be used?
The pLenti6/TR lentiviral-based vector facilitates the generation of stable cells lines that express high-levels of the tetracycline repressor (TR) under the control of the CMV promoter. Using lentiviral delivery means that this gene can be effectively delivered and efficiently integrated in hard-to-transfect, primary and non-dividing cell types. Cells expressing the TR can be selected using Blasticidin. The pLenti6/TR vector is designed for use with the BLOCK-iT Inducible H1 Lentiviral RNAi System and the ViraPower T-REx Lentiviral Expression System.

Questions about BLOCK-iT Complete Dicer RNAi Kit and BLOCK-iT Dicer RNAi Transfection Kit:

When is the best time to use an in vitro Dicing approach?
Dicer is an excellent tool to use early in RNAi analysis. It is an advanced tool for:

• Generating effective RNAi data for grant applications and journal
• An initial screen of several genes to select which are of
• Analyzing at the knockdown effect to a specific portion of
• Knocking out all splice variants or all the family members of a particular gene

Can I expect to see off target background when using d-siRNA for RNAi analysis?
It is important to take into consideration what target sequence will be used as a Dicer substrate. Dicer optimally works with long dsRNA of 500-1000 bp, although we've been successful in generating smaller siRNA duplexes (~200 bp) capable of blocking gene expression. It is important to select appropriate regions from a gene(s) of interest to amplify and transcribe. To avoid non-specific knockdown effects, avoid transcribing conserved regions and select a gene region that is specific to the gene of interest. Dicer is unique in that it also provides an opportunity to amplify conserved gene regions- and thus the expression of entire gene families can potentially be knocked out if desired.

How is Dicer different from using RNase III to cleave the dsRNA?
The RNase III enzyme apparently cleaves long dsRNA into smaller (12-15 nucleotide) fragments than Dicer. There are some reports that these resulting fragments may be effective at generating an RNAi effect, although it appears that much more product needs to be transfected to achieve this knockdown. The Dicer enzyme is a natural component of the endogenous RNAi pathway in eukaryotes, and purified recombinant Dicer used in an optimal protocol cleaves the long dsRNA to the specific size known to be effective for generating the knockdown response.

Do I have to use your BLOCK-iT TOPO Transcription Kit to generate the long dsRNA as a template to work with BLOCK-iT Dicer?
The BLOCK-iT TOPO Transcription kit is ideal for generating high yields of quality dsRNA for use as a Dicer substrate; long dsRNA from other sources will work as well.

How are the BLOCK-iT Dicer RNAi Transfection and BLOCK-iT Complete Dicer RNAi Kit configured?
Invitrogen kits are configured so that everything you need for your RNAi experiment is included, you just provide the dsRNA substrate or use the BLOCK-iT RNAi TOPO Transcription Kit (included in the BLOCK-iT Complete Dicer RNAi Kit) to generate your dsRNA.

The BLOCK-iT Dicer RNAi Transfection kit provides the necessary reagents to generate enough diced product to do up to 150 transfection experiments in 24-well plate with up to five genes. This is significantly more transfection experiments that other Dicer kits. The BLOCK-iT Dicer RNAi Transfection Kit includes:

• A Dicer Enzyme
• RNAi Purification reagents to purify the d-siRNA
• Lipofectamine 2000 for high efficiency delivery of d-siRNA to mammalian cells

If you wish to use truly optimized Invitrogen reagents to prepare your dsRNA substrate and to complete the dicing reaction, we recommend the use of the BLOCK-iT Complete Dicer RNAi. An easy to use RNAi purification module with optimized protocols for isolating the dsRNA (BLOCK-iT RNAi TOPO Transcription Kit) or d-siRNA (BLOCK-iT Dicer RNAi Transfection Kit) is included with these kits. These are critical for best results. The purity of long dsRNA can affect how well this will act as a substrate for the dicing reaction. The optimized purification of the d-siRNA is essential, since if this is not purified completely away from the long dsRNA a general cell shutdown response can be triggered.

The leading siRNA transfection reagent, Lipofectamine 2000 is also included in our BLOCK-iT Dicer RNAi Transfection Kit to assure high efficiency delivery and experimental success.

Questions about Custom RNA Oligos

What chemistry is used for synthesis of Invitrogen's RNA Oligonucleotides?
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.

What purification methods are used for RNA oligonucleotides?
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.

What are the differences between Desalted and HPLC purifications?
HPLC purification means that the oligo is purified for full-length. Desalting means that the by-products of synthesis are removed from the oligo. No purification of failure sequences is involved.

Is there a maximum length for synthesis of RNA oligonucleotides?
The current maximum length of RNA oligonucleotides is 50 bases.

What is the coupling efficiency for RNA oligonucleotides?
98.5%

Which kind of quality control is used for the RNA oligonucleotides?
Synthesis is monitored through trityl analysis. Final testing by mass spectroscopy to further ensure the quality.

What synthesis scales are offered for RNA oligonucleotides and how much RNA will I receive?
The offered synthesis scales are 50 nM and 200 nM. If the synthesis scale is 0.2 µmol the final yield will not be 200 nmol. The guaranteed yields are: Single Stranded RNA Specifications

NOTE: 1 nmole scale also available in plate format.

In which form do I receive my RNA oligonucleotides?
Lyophilized

How can I tell how much RNA I have received?
For single stranded RNA, the product documentation includes the mass, moles, and OD numbers for each shipment. For duplex the amount of product delivered is standard (see specifications) and listed on the tube the COA will still provide the details for the single strand.

Which modifications can be used for RNA?
Please reference the modifications list.

Can I get an RNA oligo with a 2 deoxyTs at the 3' end?
Yes. Simply include TT in the requested sequence.

How should RNA oligonucleotides be stored?
The best way to store RNA is as a dry pellet at -20°C or colder.

How stable is the RNA?
As a dry pellet the can be stored at -20°C for 6 months.

What should I dissolve my RNA pellet in, water or buffer?
We recommend dissolving the single stranded RNA in 1X TE buffer (prepared under RNase-free conditions(10 mM TrisCl, pH7.5, 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 water to bring the RNA conc. to 20 µM will reconstitute the buffer to the same.