FlexiTube siRNA Premix

1ステップsiRNAトランスフェクション用に至適化済み試薬

Products

FlexiTube siRNA Premixは分子生物学的アプリケーション用であり、疾病の診断、予防、あるいは治療に使用することはできません。
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FlexiTube siRNA Premix

Cat. No. / ID:  1027420

Optimized siRNA–reagent mix; 0.75 nmol siRNA; provided in tubes
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Features

  • 細胞に添加し、インキュベートするだけの簡単操作で時間を節約
  • より簡単な操作で効率的なトランスフェクション
  • 一定した条件下で多数の実験が可能

Product Details

FlexiTube siRNA PremixはsiRNAとトランスフェクション試薬が最適な割合で配合されています。siRNAと試薬が混和済みなので試薬の混和やコンプレックス形成のための操作が不要で実験時間を短縮できます。さらにFlexiTube siRNA Premixを用いれば、siRNAと試薬の比率を決定するための至適化実験の必要がありません。FlexiTube siRNA Premixはヒト、マウスの遺伝子用があります。

Performance

FlexiTube siRNA Premix中のsiRNAと試薬との比率は、高いトランスフェクション効率とノックダウン効率を示します(図 “  迅速で効率的なノックダウン”および“  ノックダウン後の表現型解析”)。

FlexiTube siRNA Premixに入っているFlexiTube siRNA はQIAGENの高性能なHP OnGurad siRNA Designを用いてデザインされ、高い抑制効果と特異性を実現します。siRNAは膨大なRNAi 実験のデータセットを基にしたニューラルネットワークテクノロジーを用いてデザインされています。その後、適正に評価された重複のない最新の配列データベースと新しい特徴を取り入れたQIAGEN独自の相同性解析ツールを用いてsiRNAデザインとゲノムの他の配列との相同性をチェックします。

性能保証

FlexiTube siRNA Premixesには1回だけ無料で再納品する保証が付いております。同じターゲット遺伝子に対して数種類のFlexiTube siRNA Premixesを注文され、そのうち少なくとも2種類が ≥70%のターゲット遺伝子のノックダウンを実現しなかった場合、QIAGEN は追加料金無しで、2種類のsiRNAを1度だけ再納品します。再納品に際しては適切なトランスフェクション条件下でsiRNAがmRNAレベルでターゲット遺伝子を最低70%ノックダウンできなかったことを証明するデータが必要です。このデータにはトランスフェクション効率、silencingの定量データ、ポジティブコントロールで70%以上のノックダウン効果を示すデータも含まれます。このオファーは製品納入後6か月まで有効です。

See figures

Principle

FlexiTube siRNA Premixは、目的遺伝子をターゲットにしたsiRNAと高効率なトランスフェクション試薬が既に混和されており、1本のチューブで供給しております。FlexiTube siRNA PremixにはsiRNA・試薬コンプレックスの安定性を高める特殊なバッファーが入っています。FlexiTube siRNA Premix中のsiRNAと試薬と の比率は至適化済みで、高いトランスフェクション効率とノックダウン効率を示します(図“  迅速で効率的なノックダウン”および“  ノックダウン後の表現型解析”)。
See figures

Procedure

FlexiTube siRNA Premixは細胞へのトランスフェクションへ即使用可能です。細胞に添加しインキュベートするだけの簡単な操作です。通常のトランスフェクション実験はsiRNAを希釈してトランスフェクション試薬と混和、これをインキュベートしてsiRNA試薬のコンプレックスを形成し、このコンプレックスを細胞に添加してインキュベーションを行なう、というステップが必要ですが、これと比較すると、FlexiTube siRNA Premixは時間と労力を大幅に節約できます。

FlexiTube siRNA Premixを用いると、siRNA と試薬の比率を至適化する必要がないのでRNAi実験をすぐに開始できます。FlexiTube siRNA PremixではsiRNA と試薬の比率が最適に混和されているため、多くのトランスフェクションを含む時間のかかる至適化実験は最小限に抑えられるか回避することができます。

1本のFlexiTube siRNA Premixで複数のトランスフェクションが行なえます。これは実験間におけるばらつきを抑え、一定した信頼できる実験結果を実現します。

Applications

FlexiTube siRNA Premixは、少数の標的遺伝子を用いた機能ゲノミクスあるいはパスウェイ解析に最適です。

Supporting data and figures

Specifications

FeaturesSpecifications
DesignHP OnGuard design
Target sequence providedYes
ModificationNo
siRNA per target genevariable
FormatTube
Number of transfections50 standard transfections in 24-well format
Guarantee/validationfor validated siRNAs
SpeciesHuman, mouse
Scale or yield0.75 nmol

Resources

テクニカルインフォメーション (1)
MSDS (1)
Download Safety Data Sheets for QIAGEN product components.
Safety Data Sheets (1)
Certificates of Analysis (1)
Gene Expression Analysis (1)

FAQ

What are the suggestions for unsuccessful gene knockdown?

Determine the transfection efficiency and identify the optimal siRNA concentration for the cell type. Assess the gene knockdown effect at mRNA level using real-time PCR. In some cases, you may need to assess mRNA levels at 48, 72, and 96 hours post-transfection. You may also want to include positive controls for both transfection and gene knockdown experiments.

If the issue persists, send real-time PCR data and/or western blot data to QIAGEN Technical Service for further assistance
FAQ ID -9031
Is one able to use FlexiTube siRNA Premix for transfection of primary adherent cell types?
QIAGEN's experience using FlexiTube siRNA PreMix and transfection of primary adherent cell types is limited, but we do know that researchers have achieved excellent results with some primary cell types, for example, HUVEC cells.
FAQ ID -2268
How do I store my FlexiTube siRNA Premix?
Reconstituted FlexiTube siRNA Premix can be stored at 4°C for one month.  For storage longer than one month, it is recommended to store the reconstituted mix at -20°C.  We found that after 5 freeze-thaw cycles, the potency of FlexiTube siRNA Premixes was not affected. However, although it is not recommended, we found that storage of reconstituted FlexiTube siRNA Premix for 6 months at 4°C did not reduce the gene silencing potency compared to reference transfections.
FAQ ID -2270
What are the critical parameters to when optimizing transfection conditions?
The following parameters may be assessed, in an effort to maximize transfection efficiencies:

The amount of siRNA/shRNA being delivered

The optimal amount will be dependent upon the cell line, and target gene, under study. For most experiments, maximal potency, with minimal off-target effects, is achieved between 1nM to 100nM siRNA or shRNA plasmid.

The amount of transfection reagent

This is dependent upon the transfection reagent being used and should be optimized carefully. For QIAGEN’s transfection reagents you will find helpful starting conditions for optimization based on real experimental data on our Transfect Protocol database (http://www.qiagen.com/transfectionprotocols/default.aspx).

Length of transfection complex formation incubation period

Many chemical transfection reagents have a “sweet spot”, at which time a transfection complex of optimal diameter is formed. This is typically between 5 to 30 minutes, depending upon the nature of the reagent. Refer to the reagent manufacturer’s recommendations.

Cell line

Optimal transfection conditions are extremely cell line-dependent. The amount of siRNA/shRNA and transfection reagent, as well as the amount of time that the transfection complex should be left on the cells, will vary from one cell line to another. Helpful information is always available at the Transfect Protocol Database (http://www.qiagen.com/transfectionprotocols/default.aspx).

Cell density

This will be cell line-dependent. For most adherent cell lines, cultures that are 60-80% confluent at the time of transfection are typically optimal. For suspension cultures, densities between 0.5-1.0 X 106 cells/ml are typically optimal.

Cell passage number

Transfection efficiency declines the longer the cells are kept in culture. It is recommended that cell cultures that have been in culture beyond 10 passages NOT be used for transfection. Always take care to make sure that the cell cultures to be transfected are actively dividing, and are at least 90% viable, prior to transfecting.

Traditional vs. reverse transfection protocol

In some instances, plating cells onto wells or plates containing transfection complexes may result in increased transfection efficiency, compared to the traditional approach of adding transfection complexes to an established culture. An additional benefit to such reverse transfection protocols is that seeding and transfecting cells on the same day shortens the experimental timeline by a full day.

Electroporator settings

When utilizing electroporation to deliver siRNA/shRNA to cells that are difficult to transfect via conventional chemical methods, the voltage, pulse length, and pulse number are three critical factors which will require optimization. For additional information, refer to your instrument’s user’s manual.
FAQ ID -9032
Is there a guarantee for the performance of FlexiTube siRNAs in gene knockdown?
Yes. FlexiTube siRNA comes with a one-time–only replacement offer. If 2 or more FlexiTube siRNAs for the same target gene are ordered and two or more do not result in gene silencing, QIAGEN will provide 2 additional siRNAs free of charge, once only. You will be asked to provide supporting data, demonstrating that the siRNA failed to knock down the target gene by at least 70% at the mRNA level under appropriate transfection conditions. Supporting data should include transfection efficiency data, quantitative silencing data, and data showing ≥ 70% knockdown of a positive control. This offer is valid for up to 6 months after the date of delivery.

 

FAQ ID -9040
Does the atypical look of the lyophilized FlexiTube siRNA Premix have a negative impact on performance?
The pellet of the lyophilized FlexiTube siRNA Premix does not look like a typical nucleic acid pellet.   The lyophilized FlexiTube siRNA Premix has a gel-like appearance.  This is the typical appearance of this product and has no impact on the quality of the material or its performance in downstream assays.
FAQ ID -2267
Do I have to order at least 4 FlexiTube siRNA Premixes?
No, there is no minimal order number for FlexiTube siRNA Premix. You can order one single FlexiTube siRNA Premix if desired.
FAQ ID -2266
What controls are important to include in a well designed RNAi experiment?

Negative controls are of critical importance, when performing RNAi studies, in order to confirm that any observed molecular and/or cellular changes are due to the sequence-specific RNAi event. Ideally you should use a scrambled artificial sequence that does not match any of the genes of the cell line/cell type being studied. It is important that appropriate experiments be carried out in advance to validate that the negative control siRNA under consideration has minimal impact on cell viability, proliferation, and global gene expression. The molar amount of negative control siRNA molecules used must be the same as the amount of experimental siRNA that are to be used in the knock down studies.AllStars Negative Control siRNA has been tested thoroughly for potential off target effects and has proven as suitable negative control siRNA already for many years.

Positive controls are also very useful, particularly when carrying out preliminary transfection optimization and/or assay development studies. As with the negative controls, positive controls should be experimentally validated in your model cell line of interest, at the appropriate siRNA concentration, prior to adopting them as acceptable controls. AllStars Cell Death Control siRNA is a phenotypic siRNA, which does not require tedious analysis steps.

 

FAQ ID -9034
What are the critical factors for reliable RNAi validation using qRT-PCR?

 

Our R&D team has carried out an extensive study aimed at answering this question. A thorough evaluation of the major contributing factors essential to RNAi validation using qRT-PCR was carried out. A complete description of this study can be found at the following web address:

 

http://www.sabiosciences.com/manuals/shRNAwhitepaper.pdf

 

The conclusion of this study was that the three most important criteria to meet, in order to establish a reliable RNAi validation protocol, are as follows:

 

• Transfection efficiencies of 80% or higher

 

• Standard deviation in the technical replicate raw Ct values from the qPCR analyses should be no greater than 0.2.

 

• Carry out the experiment with no less than three biological replicates of each target gene-specific siRNA/shRNA and each negative control siRNA/shRNA.

 

FAQ ID -9036
What kind of control should I use in my RNA interference experiments?

"QIAGEN offers a variety of positive and negative control siRNAs. In addition, any of our functionally validated FlexiTube siRNAs are suitable positive controls for RNA interference (RNAi). Our AllStars Negative Control siRNAs, a randomly designed sequence with no known homology to mammalian genes, is the most thoroughly tested and validated negative control siRNA currently available. We strongly recommend to use our RNAi Human/Mouse Starter Kit, which includes HiPerFect Transfection Reagent, Allstars Negative Control siRNA, a positive control siRNA directed against human and mouse MAPK1 (HS/Mm_MAPK1 Control siRNA), and Allstars Hs Cell Death Control siRNA, a phenotypic control siRNA that allows monitoring gene silencing effects by light microscopy." 

FAQ ID -9037
Is one able to use FlexiTube siRNA Premix for transfection of primary suspension cell types?
Similar to other chemical transfection technologies, FlexiTube siRNA Premix is not recommended for the transfection of primary suspension cell types.
FAQ ID -2269
How do I submit a siRNA order by telephone or online?

FlexiTube siRNA, FlexiTube GeneSolution, FlexiTube siRNA Premix, FlexiPlate siRNA, and GeneFamily Lists siRNAs can be ordered by catalog number over the telephone.

However, to ensure accuracy, Custom siRNA Synthesis orders should be submitted in writing. Therefore you can use the HP Custom siRNA Order Form https://www.qiagen.com/products/genesilencing/customsirna/customsirnaorder.aspx?EmailOrdering=1.

Visit the RNAi Solutions page http://www.qiagen.com/products/rnai/default.aspx?r=2714 on our homepage for access to the Online Ordering Tool, and choose the order link for your product of interest.

FAQ ID -399
If I am working with a difficult-to-transfect cell type or if I obtain only weak silencing effects what can I do?
In the case of difficult cell types or weak silencing effects, it may be helpful to increase the final siRNA concentration during transfection. This can be achieved simply by using larger amounts of FlexiTube siRNA Premix for transfection.
FAQ ID -2265
What are the most popular methods for monitoring the delivery of a siRNA/shRNA?

Fluorescently-labeled siRNA molecules have been shown to be transfected and processed in a manner that is indistinguishable from unlabeled siRNA. Therefore, these molecules serve as a powerful tool for simultaneously optimizing both siRNA transfection efficiency and the knock down of gene expression. FlexiTube siRNA and HP Custom siRNA is available at 20 nmol scale with different fluorescence labels including AlexaFluor dyes.

Quantitative gene expression analysis via a quantitative reverse transcription-PCR (qRT-PCR) assay is the gold standard for assessing the extent of gene expression knock down in an RNAi experiment. Alternative RNA detection methods, such as Northern blots, RNase protection assays, or end-point PCR, are not quantitative enough to reliably validate gene expression knock down. The RT2 qPCR Primer Assays are available for any gene in the human, mouse, or rat genome. Using these in combination with the pre-optimized RT2 SYBR Green Mastermixes, and the RT2 First Strand Kit provides the easiest, and most reliable, method for quickly evaluating the effectiveness of your gene expression knock down protocol.

Monitoring expression at the protein level via Western blot analysis, ELISA, immunofluorescence, or a functional assay is a critical step in confirming that a gene expression knock down experiment is ultimately resulting in decreased protein levels. However, it is very important to bear in mind that the kinetics of RNA knock down and protein knock down do not usually parallel one another. If the protein under study has a long half-life, then changes in protein level will take much longer to occur than changes in the RNA level. Additionally, it is important to keep in mind that the quality of any antibody-based protein detection assay is dependent upon the quality of the antibody being used.

Phenotypic change in the cells following siRNA delivery, can sometimes be a useful readout for monitoring the effectiveness of an RNAi experiment. AllStars Cell Death Control siRNA is a phenotypic siRNA that has been developed to work in virtually all cell times, as it consists of a blend of siRNAs addressing different vital pathways.

 

FAQ ID -9035
Is it possible to use lower siRNA concentrations with FlexiTube siRNA Premix?
FlexiTube siRNA Premix works with minimal siRNA concentrations. To decrease the final siRNA concentration, simply use smaller volumes of FlexiTube siRNA Premix for transfection. For example, using only half of the volume given in the protocol will lead to a final siRNA concentration of 12.5 nM. Depending on the cell type, final siRNA concentrations of down to 1 nM can lead to sufficient gene silencing.
FAQ ID -2264
Has RNAi been successful using siRNA in Zebrafish and Xenopus?

Here are examples of references that describe the inhibition of gene expression by siRNA in Xenopus and Zebrafish:

FAQ ID -400
What is the advantage of using a negative (non-silencing) control siRNA labeled with Alexa Fluor 488?

The Alexa Fluor 488 fluorophore is brighter and more photostable than other fluorescent labels. It is tolerant of pH changes within a wide range, making it very stable in living cells. For example, fuorescence microscopy of cells transfected with Alexa Fluor- and FITC-labeled siRNAs after 24 hours showed that the signal of the Alexa Fluor fluorophore was much more persistent than that of FITC. 

FAQ ID -9033
What is the most effective method for validating gene expression knock down in an RNAi experiment?

 

The most accurate method for validating RNA interference is to carry out qRT-PCR on RNA isolated from an enriched or selected population of transfected cells. When carrying out these assays, special care should be taken to insure that highly reproducible biological replicates, as well as technical replicates of the qRT-PCR analysis are performed. This will enable the reliable detection of the roughly 1.75 to 2.0 threshold cycle differences between gene-specific and negative control siRNA/ shRNA transfected cells, which are typically seen in RNAi experiments.

 

QIAGEN has performed intensive validation experiments for FlexiTube and FlexiPlate siRNAs resulting in more than 3700 experimentally tested siRNA now available at GeneGlobe.

 

FAQ ID -9038
What ought I do when working with a difficult to transfect cell type or if I obtain only weak silencing effects?

In the case of difficult cell types or weak silencing effects, it may be helpful to increase the final siRNA concentration during transfection. This can be achieved simply by using larger amounts of FlexiTube siRNA Premix for transfection.

FAQ ID -9039