FlexiPlate siRNA

柔軟性の高い経済的なRNAiスクリーニング用ツール

S_2781_GEF_FlexiPlate_s
GeneGlobeで探索・設定する
適切なターゲット特異的アッセイおよびパネルを探すか、またはターゲットをカスタムデザインし、お客様のご興味のある生物学的ターゲット評価にお使いください。
FlexiPlate siRNA, 0.1 nmol

Cat. No. / ID:  1027411

Custom siRNA set in 96-well plate format, 0.1 nmol
GeneGlobeで探索・設定する 価格を確認するには
Plate type
96-well
384-well
Quantity
0.1 nmol
1 nmol
0.25 nmol
FlexiPlate 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。
GeneGlobeで探索・設定する
適切なターゲット特異的アッセイおよびパネルを探すか、またはターゲットをカスタムデザインし、お客様のご興味のある生物学的ターゲット評価にお使いください。

特徴

  • siRNA、合成スケールが選択できプレートレイアウトも可能
  • 経済的なツールでより多くの標的遺伝子のスクリーニングを実現
  • 迅速かつ簡単なGeneGlobeポータルサイトへのアクセス
  • 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
  • 迅速な納期でスクリーニングを遅延なく実現

製品詳細

FlexiPlate siRNAは選んだ標的遺伝子を96ウェルフォーマットでは0.1 nmol、0.25 nmol、1 nmolの合成スケール、あるいは384ウェルプレートでは0.1 nmol、0.25 nmolの合成スケールから選択でき、ご要望にマッチしたRNAiスクリーニングを実現します。最大のフレキシビリティーは、GeneGlobeウェブポータルサイトでsiRNAを選択し、お客様自身でプレートレイアウトができることです。多数の遺伝子ファミリーであらかじめ選択されたsiRNAのリストが利用できます。siRNAはニューラルネットワークテクノロジーを独自のホモロジー解析に組み込んだHP OnGuard siRNA Designを用いてデザインされており、3' UTR/seed領域解析、非対称性、SNPの回避やインターフェロンモチーフの回避という優れた特徴を備えています。

パフォーマンス

最先端のsiRNAデザイン

siRNAデザインプロセスが進歩し、QIAGENの非常に革新的で洗練されたHP OnGuard siRNA Designによって、強力で特異的なsiRNAが確実に作られるようになっています。siRNAは、RNAi実験の超大量データを基に、ニューラルネットワークテクノロジーを利用してデザインされます。その後、最新の非冗長配列データベースおよび専用の相同性解析ツールを用い、他の全ゲノム配列とsiRNAデザインの相同性を確認します。HP OnGuard siRNA Designはユニークで高度な特長を取り揃えています(表参照)。

HP OnGuard siRNA Designの特長
特徴 説明 引用文献
ニューラルネットワークテクノロジー 非常に大きな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の有効性を実証したプロジェクトからのデータをデザインプロセスの強化および改善に利用。新薬の開発につながるような多数のゲノムsiRNAは、このプロジェクトにより少なくとも70%のノックダウンを行なうことが証明された。 4
ホモロジー解析 解析では、専用のツールと最新の非冗長配列データベースを利用。
Affymetrix GeneChip解析 ゲノムワイド解析がオフターゲット効果を最小限に抑えるsiRNAデザイン改良の開発を実現。
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。これにより5'末端での結合が弱いアンチセンス鎖がRISCに取り込まれ、一方センス鎖は分解される。非対称性により機能性の高いsiRNAがデザインされ、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が抑制される。 5, 6
3' UTR/seed領域解析 解析では合理的に比較検討する複数のパラメーター検索を利用し、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'非翻訳領域とのマッチを検討する(詳細は本文を参照)。 7-12
SNPの回避 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.Science 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、標的としていない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(表参照)。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。このようなマッチは、siRNAがmiRNAの作用を模倣することによる、目的としていない標的のダウンレギュレーションに関与する可能性があります。QIAGENでデザインされたsiRNAは、ヒト、ラット、マウス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されています。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のアライメントが行なわれます。

siRNA Seed領域の6ヌクレオチドのうちの6つが無関係な標的遺伝子の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1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は排除され、標的としない遺伝子に対して相同性が低い他の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全く示さないsiRNAを選択することは不可能です。これらの場合には、GeneGlobeではそのsiRNAが標的としない無関係な遺伝子のEntrezGene IDを提供しています。このタイプの相同性が観察されたという事実は、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はその後の解析で意図しない標的遺伝子へ影響することも考えられます。

原理

FlexiPlate siRNAにより、特殊な要件を満たすRNAiスクリーニング実験のデザインが可能です。ヒトまたはマウス遺伝子のsiRNAを選択でき、必要なポジティブ/ネガティブコントロールも選択できます。使いやすいウェブサイト上で、siRNAやコントロールを96ウェルプレートまたは384ウェルプレートの好きな位置に配置することも、あるいは様々な既製のレイアウトパターンからプレートレイアウトを選択することもできます。siRNAは0.1 nmol、0.25 nmol、または1 nmolのスケールから選択できるため、必要に応じて少量または大量のsiRNAを用い、経済的なスクリーニングが可能です。96ウェルプレートでは、0.1 nmol、0.25 nmol、1 nmolスケールが利用できます。384ウェルプレートでは、0.1 nmol、0.25 nmolスケールが利用できます。

操作手順

弊社ポータルサイトのGeneGlobe Webでは、目的の遺伝子を標的にしたsiRNAの検索およびスクリーニング実験に適したプレートレイアウトも簡単に行なうことができます。遺伝子名リスト、Entrez Gene ID、RefSeq ID、siRNA名、あるいはカタログ番号をアップロードできるので、簡単かつ迅速なプレート注文プロセスになっています。プレートはすぐに注文することも、まず保存しておき、後から変更したり、注文することも可能です。記録用にプレートレイアウトのダウンロードも簡単に行なうことができます。

アプリケーション

FlexiPlate siRNAは以下のようなRNAiアプリケーションに使用可能です。

  • パスウェイ解析
  • スクリーニング実験の追跡

裏付けデータと数値

Specifications

FeaturesSpecifications
DesignPredesigned/HiPerformance siRNA Design Algorithm
FormatPlate
Target sequence providedYes
ModificationNo
SpeciesHuman, mouse
Guarantee/validationNo guarantee
Scale or yield0.1 nmol, 0.25 nmol, 1 nmol

リソース

サイエンティフィック・ポスター (1)
Poster for download
テクニカルインフォメーション (1)
追加リソース (1)
MSDS (2)
Download Safety Data Sheets for QIAGEN product components.
Download Safety Data Sheets for QIAGEN product components.
キットハンドブック (1)
For transfection of eukaryotic cells with siRNA and miRNA
Safety Data Sheets (1)
Certificates of Analysis (1)

FAQ

Are FlexiPlate siRNAs available at a higher final concentration than 10 µM?

We cannot provide FlexiPlate siRNAs at a higher final concentration. To obtain more concentrated siRNA solutions, individual lyophilized siRNAs can be resuspended in smaller amounts of sterile RNase-Free water. See FAQ 1360 for additional details on recommended resuspension volumes for various scales of FlexiPlate siRNA.

FAQ ID -1476
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
If FlexiPlate siRNA was accidentally redissolved in siRNA Suspension Buffer instead of water, resulting in a 2x buffer, would it affect performance of the siRNAs?

We do not expect a difference in performance of the siRNAs in case FlexiPlate siRNA was accidentally redissolved in siRNA Suspension Buffer. However, we have not tested this and do not have any proof data.

 

FAQ ID -1477
What information is provided with FlexiPlate siRNAs?

With FlexiPlate siRNA, you receive a CD with an Excel file that contains the following information (example):

  • Plate ID: 1
  • Row: A
  • Column: 2
  • Entrez Gene ID: 9210
  • NCBI Gene Symbol: BMP15
  • Gene description: bone morphogenetic protein 15
  • siRNA target sequence (for HP GenomeWide siRNAs): TCGGAGTATAAGTATGAATTCCAA
  • RefSeq ID (mRNA accession): NM_00222
  • mRNA location: 756
  • mRNA region: CDS
  • Catalog number SIxxxxxx

 

FAQ ID -1363
I have a Human Druggable Genome siRNA Set V2.0. Can I reorder the siRNAs from this set?

We have updated GeneGlobe and have exchanged several siRNAs with new designs. To find and order siRNAs from your set in GeneGlobe, search by using the siRNA catalog number (e.g., SI00000203). If you search by entering the gene name or accession number, only the new siRNA designs will be shown.

 

FAQ ID -1366
What information is required to reorder specific siRNAs from a siRNA Set in FlexiPlate format?

Individual siRNAs from the QIAGEN HP siRNA Sets can be reordered in FlexiPlate format by entering the corresponding GeneGlobe catalog number (SI number) for the specific siRNA. SI number information for each siRNA is provided on the annotation CD which is included in the shipment of the siRNA Set.

FAQ ID -1463
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
Which controls can I choose for FlexiPlate siRNA?

You can add the following controls to your FlexiPlate siRNA plate: AllStars Negative Control siRNA, AllStars Cell Death Control siRNA, Negative Control siRNA, Human GAPDH siRNA, Human Beta-Actin siRNA, Human and mouse MAPK1 siRNA, Human or mouse Lamin A/C siRNA, Mouse AKT1 siRNA, or other siRNAs from GeneGlobe, such as HP Validated siRNAs.

FAQ ID -1368
How can I upload my gene list for FlexiPlate siRNA to GeneGlobe?

You can upload an Excel file with your gene list for FlexiPlate siRNA, including gene-specific information, to GeneGlobe. At the GeneGlobe site, an example file with details on how to enter information is provided.

 

FAQ ID -1364
Which kind of 96 and 384-well plates are available for FlexiPlate siRNAs?

FlexiPlate siRNAs will be supplied in NUNC Polystyrene V bottom plates.  NUNC Polystyrene plates can be ordered from Thermo Scientific Company.

  • Nunc MicroWell* 96-Well Microplates catalog # 249940.
  • Nunc* 384-Well Polystyrene Plates catalog # 265203.

http://www.thermoscientific.com/ecomm/servlet/home?storeId=11152&langId=-1

 

Other plates like ABgene AbGene Polypropylene plates can be used but ONLY upon request

FAQ ID -1459
How to upload a Flexiplate on QIAGEN Webpage?

From the home page go to>Products>Genes & Pathways>Plate Designer. Alternatively, Click on this link Upload an Excel File

Ordering FlexiPlate siRNA
Use this form to order FlexiPlate siRNA.
  Order Form for FlexiPlate siRNA

Along with the Word Order Form, you must also send plate layout information. If you have configured your plates on our Website, download the plate data and send it with the Order Form. Alternatively, you can use the Excel template below to create your own spreadsheet. For help filling in the template, see our detailed siRNA plate template instructions.

You can also use this template to create your own file for uploading to our Website, where you can configure and order your plates online.


*     Excel Template (CSV version)

FAQ ID -3103
What is the composition of FlexiPlate siRNA buffer?

FlexiPlate siRNA buffer is 1x annealing buffer:

  • 30 mM HEPES (pH 7.4)
  • 100 mM Potassium Acetate
  • 2 mM Magnesium Acetate

 

FAQ ID -1361
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
Which formats are available for FlexiPlate siRNA?

FlexiPlate siRNA is available in 3 different amounts: 0.1 nmol, 0.25 nmol, or 1 nmol per siRNA. Up to 4 human or mouse siRNAs can be selected per gene.

FlexiPlate siRNA is provided lyophilized in 96-well plates and is shipped at room temperature. siRNAs can be arranged in plates using the drag-and-drop tool at the GeneGlobe Web portal. Special formats are available on request: delivery in 384-well plates, delivery in solution and aliquoting.

 

FAQ ID -1358
Can FlexiPlate siRNA be ordered in solution on dry-ice?

We strongly recommend our standard method of shipment for FlexiPlate siRNAs in lyophilized form at room temperature. Delivery in solution on dry ice is possible as an exception only and has to be requested specifically when placing the order.

FAQ ID -1474
Can FlexiPlate siRNAs be ordered in plates other than the standard NUNC 96 well V bottom plates?

We do not provide FlexiPlate siRNAs in customer-specific plates for single plate orders. For large orders, there is the possibility to switch to other plates. Please inquire for details by contacting QIAGEN Technical Service.

 

FAQ ID -1458
How many transfections can be performed with Flexiplate siRNAs?

0.1 nmol scale – add 10 ul of 10uM stock solution.

120 transfections in 96 well plates at 5 nM final concentration OR

60 transfections at 10nM final concentration.

 

0.25 nmol scale - add 25 ul of 10uM stock solution.

300 transfections in 96 well plates at 5 nM final concentration OR 

150 transfections at 10nM final concentration.

 

1.0 nmol scale - add 50 ul of 20uM stock solution.

1200 transfections in 96 well plates at 5 nM final concentration OR 

 600 transfections at 10nM final concentration.

FAQ ID -3105
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
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
Can I order Alexa Fluor labeled controls with FlexiPlate siRNA?

Labeled siRNAs must be ordered separately. They cannot be provided on the same plate with FlexiPlate siRNA.

 

FAQ ID -1369
What is the minimum number of siRNAs per order? If I order 40 siRNAs, can I get those on 2 plates?

The minimum order number is 36 in 96 well plates or on average 36 siRNAs per plate. The minimum average number of siRNAs per plate is 36. Thus, with 40 siRNAs, you would receive the siRNAs on one plate. For 72 siRNAs, you can split the siRNAs on 2 plates with 36 siRNA on each plate. If you order 150 siRNAs, you can for example get 3 plates with 50 siRNAs each.

 

To summarize: The minimum number of siRNAs for each catalog Number (or siRNA Sacle -1 nmol, 0.25 nmol or 0.1 nmol) is 36. Within a specific scale, plate can have less than 36 siRNAs, if the minimum average number of siRNAs per plate is 36 siRNAs.

FAQ ID -1468
If I cannot order FlexiPlate siRNA online, how do I order?

You can search for FlexiPlate siRNAs and design your plate layout online and then download it to an Excel file. Send the Excel file or the ID number of the Excel file to QIAGEN by fax or e-mail. Please also send the Word Flexiplate siRNA Order Form which includes shipping and billing information. Find out more about how to order if you cannot order online.

 

FAQ ID -1371
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
How do I resuspend my FlexiPlate siRNAs?

FlexiPlate siRNAs are preannealed and dried down from a 10 µM solution in buffer. They should be resuspended in sterile RNase-free water only (you do not need siRNA buffer to resuspend the siRNAs).

To achieve a final siRNA stock concentration of 10 µM, resuspend:

  • 0.1 nmol siRNA in 10 µl
  • 0.25 nmol siRNA in 25 µl
  • 1 nmol siRNA in 100 µl

Mix gently and cover the plate with new sealing film. Allow siRNAs to dissolve for 30 minutes at 4°C with occasional shaking or gentle vortexing.

 

FAQ ID -1360
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
Can different amounts of siRNA be ordered on one FlexiPlate?

Only one scale of siRNAs is available per FlexiPlate. If different scales are required, siRNAs have to be ordered on separate FlexiPlates. When ordering different scales on different plates, note that the average number of siRNAs per plate has to be a minimum of 36.

Please see FAQ 1370 for an example and further details.

FAQ ID -1473
What are the turnaround times for FlexiPlate siRNA orders?

The standard turnaround time for FlexiPlate siRNA orders is 8 to 10 days. Turnaround times for special orders requiring pooling or aliquotting will differ, and will be communicated to the enduser upon ordering.

FAQ ID -1478
What information about my gene or siRNA do I need to order FlexiPlate siRNA?

To locate and order specific FlexiPlate siRNAs you can upload a list of search terms such as:

  • Entrez Gene IDs (e.g., 100)
  • Gene symbols (e.g., ADA)
  • RefSeq IDs (e.g., NM_000022)
  • Catalog numbers (e.g., SI00000203)
  • siRNA names (e.g., Hs_ADA_1)
FAQ ID -1365
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 standard format for Flexiplate siRNAs delivery?

Flexiplate siRNAs are delivered annealed and lyophilized.  Each plate  is accompanied by an instruction sheet detailing how to reconstitute the siRNAs in the appropriate volume of RNase-free water, as well as a CD containing the siRNAs IDs, sequences information and gene annotations. See FAQ 1363.

FAQ ID -3104
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