FlexiTube siRNA

1遺伝子の効率的なRNAi同時解析用

Products

FlexiTube siRNA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。
Image
FlexiTube GeneSolution

Cat. No. / ID:  1027416

siRNA GeneSolution details
Copy order details
Image
FlexiTube siRNA (20 nmol)

Cat. No. / ID:  1027419

20 nmol siRNA with modification options delivered in tubes
Copy order details
Image
FlexiTube siRNA (20 nmol)

Cat. No. / ID:  1027418

20 nmol siRNA delivered in tubes
Copy order details
Image
FlexiTube siRNA (5 nmol)

Cat. No. / ID:  1027417

5 nmol siRNA delivered in tubes
Copy order details
Image
FlexiTube siRNA (1 nmol)

Cat. No. / ID:  1027415

1 nmol siRNA delivered in tubes
Copy order details

Features

  • 経済的なsiRNAにより多くの遺伝子解析が可能
  • どの遺伝子にも対応する最適なRNAi ソリューション
  • 最新のsiRNAデザインでオフターゲット効果のリスクを最小限に抑制
  • GeneGlobeポータルサイトでsiRNAを簡単に検索/注文

Product Details

FlexiTube siRNAには、FlexiTube siRNA およびFlexiTube GeneSolutionの2つの異なる製品がございます。

FlexiTube siRNAにより少数の遺伝子を対象としたRNAi解析実験を経済的に行なえます。ヒト、マウス、ラットのsiRNAでは 5 nmolあるいは20 nmolで、ヒト、マウスのsiRNAでは1 nmolの経済的な合成スケールが入手可能です。

FlexiTube GeneSolutionは1標的遺伝子当たり4種類のsiRNA(1 nmol)をセットにした遺伝子特異的な製品です。FlexiTube GeneSolution を使用することで、複数のsiRNA による信頼性の高い検証実験が効果的に行なえます。革新的なHP OnGuard siRNA Design を用いてデザインされたFlexiTube siRNA およびFlexiTube GeneSolutionは、QIAGENのGeneGlobeポータルサイトから入手可能です。

Performance

性能保証

FlexiTube siRNA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 siRNAsを注文され、そのうち少なくとも2種類がターゲット遺伝子を 70%以上ノックダウンしなかった場合、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。

実験的に検証済みの数千のsiRNA

数千のヒトsiRNAにおいて、リアルタイムRT-PCR 解析で70%以上のノックダウン効果のあることが実証済みです。これらの検証済みsiRNAを使用した実験の詳細やノックダウンレベルの情報は GeneGlobeでご覧いただけます。これらのsiRNAは、QIAGENの研究者により行なわれた世界最大規模のsiRNAバリデーションプロジェクトの結果です。このプロジェクトに関する詳細は論文Krueger, U. et al. (2007) Insights into Effective RNAi Gained from Large-Scale siRNA Validation Screening. Oligonucleotides 17, 237をご覧ください。

最先端のsiRNAデザイン

siRNAデザインプロセスにおける進歩により、QIAGENの高性能なHP OnGurard siRNA Designは効果的かつ特異的なsiRNAを確実にお届けします。膨大なRNAi実験のデータセットをもとにしたニューラル·ネットワークテクノロジーを用いてsiRNAをデザインしています。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。HP OnGuard siRNA Designは以下の特長を取り揃えています(表1)。

HP OnGuard siRNA Designの特長
特長 概要 参考文献
ニューラル・ネットワークテクノロジー 大規模なRNAiデータセットをベースにしたBioPredsi neural networkをsiRNAデザインに利用 1-3
世界最大規模のsiRNA 検証プロジェクト QIAGEN研究者が数千のsiRNAのノックダウン効果を実証したプロジェクトからのデータにより、デザインプロセスは強化および改善された。多数の創薬ゲノムsiRNAが、このプロジェクトで少なくとも70%のノックダウンを提供することが証明されている。 4
ホモロジー解析 解析は独自のツールと重複のない最新の配列データベースを利用
Affymetrix GeneChip解析 ゲノムワイド解析によりオフターゲット効果を最小限に抑えるsiRNAデザインの改良開発を実現
最新のsiRNA標的配列 NCBIデータベースからの最新データにより正確なデザインを実現
非相称 siRNA は5'末端塩基対の安定性が同等ではないようにデザインされている。5'末端の結合が弱いアンチセンス差がRISCに取り込まれ、一方センス鎖は分解される。非相称は、機能性の高いsiRNAをデザインし、センス鎖がRISCに組み込まれて起こるオフターゲット効果のリスクを抑制する。 5, 6
3' UTR/Seed領域 解析は、siRNAアンチセンス鎖のSeed領域と目的としていないmRNA標的の3'非翻訳領域との一致を複数のパラメーター検索で情報処理を行なう(詳細は本文参照) 7-12
SNP回避 SNPs(single nucleotide polymorphisms)にかかるsiRNAを排除するためにRefSNPデータベースを使用。ある1つのSNPにしか対応していないsiRNAはその効果が変動するために、このプロセスによりsiRNAのサイレンシング効果が増大する
インターフェロンモチーフの回避 インターフェロン応答を起こすことが判明している複数の配列モチーフに対してsiRNAを検索し、そのようなモチーフを持つsiRNAを排除 13, 14
1.Huesken, D. et al. (2005) Design of a genome-wide siRNA library using an artificial neural network. Nat. Biotechnol. 23, 995.
2.Mukherji, M. et al. (2006) Genome-wide functional analysis of human cell-cycle regulators. Proc. Natl. Acad. Sci. 103, 14819.
3.Matveeva, O. et al. (2007) Comparison of approaches for rational siRNA design leading to a new efficient and transparent method. Nucleic Acids Res. 35, e63.
4.Krueger, U. et al. (2007) Insights into effective RNAi gained from large-scale siRNA validation screening. Oligonucleotides 17, 237.
5.Aza-Blanc, P. et al. (2003) Identification of modulators of TRAIL-induced apoptosis via RNAi-based phenotypic screening. Mol. Cell 12, 627.
6.Schwarz, D.S. et al. (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199.
7.Farh, K.K. et al.(2005) The widespread impact of mammalian microRNAs on mRNA repression and evolution.310, 1817.
8.Grimson, A. et al.(2007) MicroRNA targeting specificity in mammals: determinants beyond seed pairing.Mol.Cell 27, 91.
9.Jackson, A.L. et al.(2003) Expression profiling reveals off-target gene regulation by RNAi.Nat. Biotechnol.21, 635。
10.Lewis, B.P., Burge, C.B., and Bartel, D.P.(2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.Cell 120, 15.
11.Lim, L.P. et al.(2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.Nature 433, 769.
12.Saxena, S., Jónsson, Z.O., and Dutta, A. (2003) Small RNAs with imperfect match to endogenous mRNA repress translation.Implications for off-target activity of small inhibitory RNA in mammalian cells.J. Biol.Chem.278, 44312.
13.Judge, A.D., Sood, V., Shaw, J.R., Fang, D., McClintock, K., and MacLachlan, I. (2005) Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA.Nat Biotechnol.23, 457.
14.Hornung, V. et al.(2005) Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7.Nat Med.11, 263. 

3' UTR/Seed領域解析

いくつかの研究は、意図していないmRNAの3' 非翻訳領域(UTR)とsiRNAアンチセンス鎖のSeed領域の一致がオフターゲット効果を引き起こしていることを示唆しました(表)。Seed領域は二本鎖siRNAのアンチセンスsiRNA鎖の2~7塩基目にある6ヌクレオチドから構成されています。これらのヌクレオチドのマッチは、siRNAがmiRNAに類似したメカニズムにより標的としない遺伝子の抑制に寄与することがあります。QIAGENでデザインしたsiRNAは、ヒト、ラット、マウスのRefSeqデータベースに由来する独自の3' UTR配列セットを用いて、3' UTR/Seed領域の相補性を解析しています。miRNAに類似するオフターゲット効果に関与する可能性のある全てのホモロジーをチェックするために、これらの配列と各siRNA のアライメントを行なっています。

siRNA Seed領域の6ヌクレオチドのうちの6つが無関係な遺伝子の3' UTR配列とマッチしていることが頻繁にありますが、このようなマッチを示すsiRNAを排除することは不要ですし、実用的でもありません。Seed領域と他の相同性を示す10あるいはそれ以上の塩基とのマッチが一つのsiRNA配列内に認められることは、非常に珍しいことです。このような相同性は、オフターゲット効果を引き起こす可能性がより高いので、これらのsiRNAは取り入れられず、無関係な標的遺伝子に対して有意な相同性が低い他のsiRNAが選択されます。

いくつかの標的遺伝子では、このような相同性をまったく示さないsiRNAを選択することは不可能です。これらの場合には、そのsiRNAが目的としない無関係な遺伝子のEntrezGene IDsをGeneGlobeで提供しています。このタイプの相同性が観察されたということは、これらの遺伝子が必ずしもそのsiRNAにより影響を受けるということではありません。しかし、これらのsiRNAは続く解析で意図しない標的遺伝子へ影響することが考えられます。

Principle

FlexiTube siRNAはわずか1 nmolの経済的な合成スケールで少数のsiRNAを(ご注文は4本から)、あるいは 5 nmolおよび20 nmolでご注文いただけます。凍結乾燥のsiRNAをチューブでお届けします。

標的遺伝子の最適なsiRNAセットFlexiTube GeneSolution

関心のある遺伝子(ヒトあるいはマウス)の情報詳細をGeneGlobeポータルサイトに入力するだけで、お客様の標的遺伝子に対する効率的なRNAi ソリューションが提示されます。検索結果は、各遺伝子に対応する4種類の推奨siRNAがFlexiTube GeneSolution(1 nmol)として示されます。公表されているガイドラインでは、RNAi実験結果を確実にするために複数の実験を行なうことが推奨されています(Echeverri, C.J. et al.(2006) Minimizing the risk of reporting false positives in large-scale RNAi screens.Nat. Methods.3, 777; Echeverri, C.J. and Perrimon, N. (2006) High-throughput RNAi screening in cultured cells: a user’s guide.Nature Reviews Genetics 7, 373.)。重複実験では、オフターゲット効果を排除するため同じmRNA 上の異なる領域を標的にしたいくつかの個別のsiRNA を用います。オフターゲットによる表現型は、あるsiRNAが本来の標的転写物だけではなく他の転写物を同時に抑制した際の特異的なパターンにより誘導されます。この現象はsiRNAの配列に直接由来するため、配列の異なる複数種類のsiRNAが同じ配列に起因するオフターゲット効果を引き起こす確率は非常に低くなります。ある表現型を数種類の異なるsiRNAで確認することは、siRNAの特異性を示すために容易に実践できる簡便な方法です。FlexiPlateTube GeneSolutionを用いてヒトおよびマウスの遺伝子に対する複数のRNAi実験が行なえます。お客様の実験に最適なソリューションを直ぐに提供できるように、QIAGENでは各遺伝子に対応する適切なsiRNAをお届けしています。

siRNAの配列情報を添付

全てのsiRNAには無料でsiRNA配列情報が付いています。siRNAの配列を実験結果の解析およびノックダウンの検証のために使用できます。研究論文にsiRNA配列が必要な場合には、掲載することもできます。

修飾オプション

20 nmol スケールのFlexiTube siRNAでは、Alexa Fluor、フルオレセイン、ローダミン、Cy3、Cyanine 670 による標識、アミノ、チオ・リンカーなどの修飾オプションやリン酸修飾が可能です。

Procedure

ロースループットのsiRNAトランスフェクションにはHiPerFect Transfection Reagent、ハイスループットのsiRNAトランスフェクションにはHiPerFect HTS Reagentをお奨めします。

Applications

FlexiTube siRNAおよびFlexiTube GeneSolution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。

Resources

サイエンティフィック・ポスター (1)
Poster for download
技術情報 (1)
Extensively characterized controls for RNAi in human, mouse, and rat
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.
Certificates of Analysis (1)
Gene Expression Analysis (1)
Articles (1)
Extensively characterized controls for RNAi in human, mouse, and rat
Scientific Posters (1)
Poster for download

FAQ

How do I calculate the percentage of silencing with real-time RT-PCR for siRNA?

Please find a detailed description for the calculation of the silencing effect in QIAGEN News article 2006 e14 'Real-time RT-PCR for analysis of gene knockdown by RNAi - controls and calculations'.

 

FAQ ID -498
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
What is the difference between the 1 nmol FlexiTube and FlexiTube Gene Solution siRNAs?

The siRNA sequences are the same.  With FlexiTube siRNA 1 nmol scale, minimum 4 siRNAs required. It can be any human or mouse, any target, 4  different siRNAs, or the same siRNA.   Whereas 1 nmol FlexiTube GeneSolution is a gene-specific package of 4 pre-selected siRNAs for the same target. 

FAQ ID -3174
How long is fluorescence detectable in cells after transfection with fluorescently labeled siRNAs?

Cells transfected with Alexa-Fluor labeled siRNA still show detectable fluorescence 72 hours after transfection. Certain Alexa dyes, e.g. Alexa Fluor 546, are detectable up to one week after transfection. By comparison, when labeling siRNA with Rhodamine or Fluorescein, transfected cells should be monitored for transfection efficiency after 3-4 hours.

Since Alexa Fluor dyes are more photostable, more resistant to variable pH conditions while in transit through the cell, and much brighter than traditionally used fluorescent dyes, Alexa Fluor labeled HPP Grade siRNA is the ideal choice for monitoring transfection efficiency.

For data and additional details on using fluorescently labeled siRNA, refer to QIAGEN News article e20, 2004: 'Alexa Fluor labeled siRNA is highly effective for monitoring transfection efficiency'.

FAQ ID -392
What are the critical factors in designing the siRNA molecules to be used for RNAi studies?
The algorithm utilized to design the siRNA of interest must assure both exquisite gene-specificity (to minimize off-target effects) and potent efficacy (expression knock down). A minimum of two siRNA molecules must be used for each gene of interest, in order to confirm the gene specificity of any observed changes.
FAQ ID -2759
What is the difference between various FlexiTube siRNAs listed for the same target gene, and which one should I choose?

Different Flexitube siRNAs for the same target gene vary slightly in their nucleotide sequence and exact position along the target. All of them were selected by the unique HiPerformance siRNA Design Algorithm and thus have a very high likelihood for knocking down the target gene efficiently. Knockdown achieved with siRNA designed by the HiPerformance algorithm is usually greater than 70%.

Consequently, any of the FlexiTube siRNAs listed in GeneGlobe for a given gene can be chosen for knockdown experiments. We strongly recommend to try at least two siRNAs for independent confirmation that the observed effects are due to specific knockdown of the targeted gene.

Flexitube siRNA comes with a one-time–only replacement offer. If 2 or more HP GenomeWide siRNAs for the same target gene are ordered and none of the siRNAs result in gene silencing, QIAGEN will provide 2 additional siRNAs free of charge, once only. Please visit our website, or contact QIAGEN Technical Services for details and required proof data.

FAQ ID -1206
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
Do I need to resuspend FlexiTube siRNAs in siRNA Resuspension buffer?

You just need to add sterile RNase-free water (which is supplied with your siRNAs) to resuspend your FlexiTube siRNA. You do not need to perform incubation steps at 96°C or at 37°C. Your siRNA comes preannealed and does not need any further preparation prior to use.

 

FAQ ID -1659
Can I still reorder siRNA that has been removed from the GeneGlobe data base?

Some siRNAs have been deleted from our standard GeneGlobe offering. For those siRNAs, the entries in public databases may have changed and the transcript is no longer listed in those databases. In other cases, we have added siRNAs with a new design.

You can still find previously listed siRNAs in GeneGlobe by searching for the specific SI number (ordering number). See also FAQ 1366 on this topic.

 

FAQ ID -1668
Do I need to anneal, deprotect or desalt my QIAGEN siRNA?

QIAGEN siRNA is delivered as a stable, ready-to-use duplex and does not need to be deprotected, desalted, quantified, or annealed before use. Simply resuspend the lyophilized RNA in the sterile RNAse-free water provided and transfect. Instructions for preparing your siRNA are provided on the data sheet supplied with each siRNA shipment.

FAQ ID -398
What criteria should one use in choosing between siRNA versus shRNA for their studies?

This decision is based on two key factors – the “transfectability” of the target cells, and the desired duration of the experiment.

siRNA molecules work well for high throughput, transient studies, with cells that are easily transfected. The limitations of using siRNA are two-fold. First of all, they do not work well with a few cell types that are extremely difficult to transfect. In addition, their use is restricted to experiments studying the impact of transient suppression of gene expression.

shRNA are carried within the context of a plasmid or viral-based vector, they can be engineered to carry a reporter gene. A reporter gene provides a straightforward readout, for carrying out transfection optimization studies. In addition, a fluorescent reporter gene (such as GFP) allows the use of fluorescence-activated cell sorting (FACS) to enrich for transfected cells. Alternatively, the vector may carry an antibiotic-resistance gene, which permits the selection of a stably transfected cell population. Another obvious advantage to using a vector-based shRNA construct is that it provides a renewable source of reagent for subsequent RNAi studies.

The key benefit of using viral-based shRNA delivery vectors is that they can efficiently deliver the shRNA into cells that are difficult (or impossible) to transfect. Additionally, viral transduction is a much more efficient process than transfection.

FAQ ID -2771
Are Northern Blots sensitive enough to detect siRNA-induced gene silencing?

Yes, Northern Blot Analysis has been shown in the literature to detect siRNA-induced reduction of specific mRNA. Whether a Northern Blot will be sensitive enough to detect a mRNA under investigation mainly depends on the expression level of the respective gene in the untreated control.

You can find an example for this application in the reference "Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems". Caplen et al., PNAS 2001, vol. 98, no. 17, pages 9742-9747.

We recommend to perform real-time RT-PCR for exact quantification of mRNA expression levels.

FAQ ID -403
Can HP Validated siRNAs be ordered on the FlexiPlate?

Yes, you can order HP GenomeWide siRNAs and HP Validated siRNAs with the Flexiplate siRNA format. There is no price difference between HP GenomeWide siRNAs and HP Validated siRNAs ordered as FlexiPlate siRNA. For HP Validated siRNAs, no sequence information is provided.

 

FAQ ID -1367
Do you have a protocol for the fixation of cells transfected with fluorescently labeled siRNA?

For the fixation of cells transfected with fluorescently labeled siRNA, we would suggest to perform the following protocol:

  1. After transfection, remove the medium from the cells and wash the cells once with PBS.
  2. Incubate the cells for 15 minutes at room temperature with 4% Paraformaldehyde (in PBS, pH 7.0). The cells should be completely covered by this solution (e.g., for a 96-well plate use 50 µl solution/well)
  3. Wash the cells with PBS.

Fixed cells can be stored at 4°C for a few days.

(Note: It is also possible to use chamber slides or object slides for this procedure. Object slides should be coated to provide better growing conditions for cells. Cells can be fixed as described above and then overlayed with embedding medium to allow investigation using a fluorescence microscope. Optimal conditions for this method need to be determined by the user)

 

FAQ ID -793
Which siRNA controls are available in FlexiTube format?
siRNA Controls available in FlexiTube format are:

Mm/Hs_MAPK1 control siRNA (5 nmol)    cat. no. 1022564

GFP-22 siRNA (5 nmol)    1022064

Luciferase GL2 siRNA (5 nmol)     1022070

Luciferase GL3 siRNA (5 nmol)     1022073

Negative Control siRNA (5 nmol)      1022076

AllStars Negative Control siRNA (5 nmol)     1027280

AllStars Neg. Control siRNA (20 nmol)        1027281

AllStars Neg. siRNA AF 488 (5 nmol)         1027284

AllStars Neg. siRNA AF 546 (5 nmol)         1027285

AllStars Neg. siRNA AF 555 (5 nmol)         1027286

AllStars Neg. siRNA AF 647 (5 nmol)          1027287

AllStars Neg. siRNA AF 488 (20 nmol)         1027292

AllStars Neg. siRNA AF 546 (20 nmol)          1027293

AllStars Neg. siRNA AF 555 (20 nmol)          1027294
 
AllStars Neg. siRNA AF 647 (20 nmol)          1027295

AllStars Hs Cell Death Control siRNA (5 nmol)     1027298

AllStars Hs Cell Death Control siRNA  (20 nmol)    1027299

Negative Control siRNA (20 nmol)                 1027310

Mm/Hs_MAPK1 control siRNA (20 nmol)          1027321







FAQ ID -1661
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
Are FlexiTube siRNA and FlexiTube GeneSolution also available for rat siRNAs?

 FlexiTube siRNA is available for human, mouse and rat genes at 5 nmol and 20 nmol scale. FlexiTube GeneSolution and FlexiTube siRNA at 1 nmol scale are available for human and mouse genes only

FAQ ID -1664
Can FlexiTube siRNA or FlexiTube GeneSolution products be ordered in solution?

No, FlexiTube siRNA and FlexiTube GeneSolution products are only shipped lyophilized at room temperature. We do not offer these products in a different format.

 

 

FAQ ID -1667
What are the structures of the siRNA molecules used in RNAi studies?
The nature of the molecules used in RNAi studies fall into two classes. The first is chemically synthesized siRNA. Typically, these are 21 nt dsRNA molecules with phosphorylated 5’ ends and 3’ dinucleotide overhangs, which directly function as templates for RISC formation. These molecules are introduced via chemical or electroporation-based transfection, and mediate a transient suppression of gene expression. The second class of molecules are shRNA (short hairpin RNA). These are typically delivered to the cell as part of a plasmid or viral vector. The shRNA molecules induce an RNAi effect by serving as substrates for the cytoplasmic RNase, Dicer.
FAQ ID -2760
Can I select HP Validated and HP GenomeWide siRNAs for FlexiTube?

Yes, you can select from HP Validated and HP GenomeWide siRNAs for the FlexiTube format. There is no price difference.

 

FAQ ID -1658
Where can I find QIAGEN products for a specific gene or gene product?
You can search for specific gene products in the QIAGEN GeneGlobe Database. This easy-to-use, comprehensive Web portal allows you to find information about, search for, and order high-quality products for human, mouse, and rat genes. QIAGEN provides a vast range of gene-specific products covering every aspect of an experiment, from gene silencing to expression analysis at the mRNA or protein level.
FAQ ID -803
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
Do you have information on in vivo RNA interference experiments?

Yes. Please see the QIAGEN News Article (2005 e3) "RNAi - a promising tool for target validation studies and therapeutics" to learn about the most recent advances in this field. It provides numerous references for the aspects of in vivo siRNA delivery and siRNA stability as well as in vivo RNAi in general. QIAGEN offers economical, high-purity siRNA compatible with in vivo animal delivery systems for reliable World-class RNAi solutions.

 

 

FAQ ID -817
Can the MAPK1 control siRNA be used for rat cells?
No. The Mm/Hs_MAPK1 control siRNA is specific for mouse and human MAPK1 only. Please visit GeneGlobe to search for all currently available gene-specific products for RNAi.
FAQ ID -547
Is sequence information for HP Validated siRNAs provided in the FlexiTube format?

Yes, sequence information for FlexiTube is provided both for HP GenomeWide siRNAs and HP Validated siRNAs, in the enclosed paper documentation upon delivery.

 

 

 

FAQ ID -1665
Are your FlexiTube siRNAs preferentially designed to any particular region of the target gene?

No, the design of Flexitube siRNAs in GeneGlobe is not biased towards either the 3' or the 5' end of the target gene.

A number of siRNAs are located in the 3' untranslated region for optimized specificity of the siRNA sequences to their target gene. Since there are homologous sequences in the coding regions of other genes for many targets, it is usually easier to find highly unique siRNA sequences in the untranslated regions. Such design ensures highly specific silencing effects for the gene of interest. 

FAQ ID -1091
What is the most reliable transfection reagent for delivering shRNA plasmids and siRNA to cells in culture?

HiPerFect Transfection Reagent is optimized for siRNA transfections and enables effective siRNA uptake and efficient release of siRNA inside cells, resulting in high gene knockdown even when using low siRNA concentrations. For high-throughput siRNA screenings HiPerFect HTS Reagent is a fast and effective newcomer specifically designed to focus on robustness and cost efficiency. For the transfection of shRNA.

Attractene Transfection Reagent should be used for the transfection of shRNA (short-hairpin RNA) vectors for gene silencing experiments to achieve high efficiency. Ease and flexibility of handling enables preparation and storage of transfection complexes making Attractene Reagent suitable for use with automated systems.

http://www.sabiosciences.com/reversetransfection.php

FAQ ID -2777
Is FlexiTube GeneSolution available for other scales than the 1 nmol scale?
FlexiTube GeneSolution is only available at the 1 nmol scale in tubes.
FAQ ID -1666
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
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 are the sequences of the FlexiTube siRNAs?

You will receive the sequences of the FlexiTube siRNAs on the data sheet along with your order.

A graphic representation of the position of HP GenomeWide siRNAs along the target sequence is available for numerous genes on our website. It can be accessed via GeneGlobe after pulling up the target gene of interest, clicking on the 'Gene Symbol' link for the species of interest, and then clicking the link under 'Product name' for details on the gene product of interest.



FAQ ID -851
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