Validation Highlights and Progress

Proteintech is a pioneer in implementing genetic knockdown and knockout to validate their antibodies. They were the first in the industry to actively test their antibodies by this approach and were honored by CiteAb’s “Most exciting antibody validation initiative” award in 2016.

 

Proteintech understands that validation is a continual process. Since implementing knockdown and knockout validation, Proteintech has continued to lead the industry in target coverage validated by this method with over 2500+ targets and counting.

抗体特異性は、複数の独立した文献において研究者にとって大きな懸念材料であると述べられており(1~3)、ユーザーが新たな抗体メーカーを選択する際に最重要視されています(4)。


RNA干渉(RNAi)は、ノックダウン動物モデルを用いる以外で、抗体特異性の最も信頼のおける試験法ですが、現在のところ抗体産業における日常的な試験には適用されていません。プロテインテックによるRNAi試験法は、今後すべての新製品に対して実施される予定で、すでにいくつかの段階の現行製品へ遡及的に適用されています。

RNAi実験を展開することで、研究者が抗体実験に貴重な時間とリソースを費やす前段階において、気軽に利用できる抗体検証のゴールドスタンダードを樹立します。当社のこの試みは、新たなベンチマークを定め、おそらく市販抗体の検証基準を全体的に向上させるというさらなる意味合いを持つでしょう。 

プロテインテック製品のsiRNA検証例

(左図)AKT1 のsiRNA検証結果、緑:チューブリン、赤:AKT1(カタログ番号:10176-2-AP)、Eva Martinez-Balibrea博士提供

(右図)SDS-PAGE後、TARDBP/ TDP-43抗体(カタログ番号:10782-2-AP、希釈倍率:1:1000)を用いてウェスタンブロットを実施したA549細胞(コントロールおよびTDP-43のshRNA処理)

 

検証に使用した抗体:

販売中の1,000種類以上の抗体は、siRNA処理サンプルで検証を実施しています。

ターゲット名 カタログ番号 アプリケーション 抗体タイプ
AKT antibody 10176-2-AP ELISA,WB,IHC,IP,FC Rabbit poly
TDP-43 antibody 10782-2-AP ELISA,IF,WB,IHC,IP,FC Rabbit poly

データシートにこのアイコンがある製品は、siRNA処理サンプルによる検証を実施しています。


参考文献

(1) Couchman JR. Commercial antibodies: the good, bad and really ugly. J Histochem Cytochem.2009;15(1):7-8

(2) Kalyuzhny AE. The dark side of the immunohistochemical moon:industry. J Histochem Cytochem.2009;15(12):1099-1101.

(3) Pradidarcheep W, Labruyere WT, Dabhoiwala NF,Lamers WH.Lack of specificity of commercially available antisera:better specifications needed.J Histochem Cytochem.2008;15(12)1099-111

(4) Christi Bird. Antibody user survey. The Scientist magazine.2012 May 1, www.the-scientist.com

 

Excerpted from F1000 paper: “siRNA knockdown validation 101: Incorporating negative controls in antibody research” (PMID: 26998240)


siRNA knockdown validation 101: Incorporating negative controls in antibody research

By Dr. W Olds and Dr. J Li

Abstract

More antibody validation protocols would identify non-specific reagents – a major source of irreproducible research – with the inclusion of negative controls. This article presents an overview of one such method: siRNA knockdown. The authors outline a general protocol, the knockdown mechanism, and tips for evaluating knockdown experiments.

Keywords

knockdown, siRNA, negative control, specificity, Western Blot, shRNA, transfection, vector

Introduction

Poor quality, unverified antibodies are a major factor underlying the crisis of reproducibility in research1, resulting in an estimated $800 million of waste each year2 and a ten-fold spike in retractions over the past decade3. Behind those numbers are researchers with mounting frustration each time a new batch of reagents fails to reproduce their previous work. If we as a scientific community are to eliminate this problem plaguing our research, we must develop new and more thorough validation methods to fortify and standardize routine studies.

We invite industry to respond to the need for more rigorous validation by incorporating negative controls, an essential part of experimental design that has nonetheless been overlooked historically by antibody manufacturers and distributors. Companies typically validate with Western blots but seldom test routinely whether antibodies still produce a signal when the target protein is suppressed or removed.

Short interfering RNA (siRNA) knockdown is a technology that makes routine use of this strategy more feasible. This technology degrades target messenger RNA to ‘knock down’ the production of a protein in the cell. The combination of siRNA-treated cells and a specific antibody will result in a significant drop in signal compared to an untreated sample by Western blot (Figure 1).

Figure 1. siRNA result of AKT1 antibody (10176-2-AP) with siRNA control, HEK293 control and siRNA AKT1.

siRNA knockdown has not been the only validation initiative put forward to identify non-specific antibodies4. CRISPR and other gene editing methods knock out a gene from DNA, preventing the associated protein from ever being produced. By default, an antibody binding to any protein in this environment is binding to the wrong protein. Knock-out is a powerful negative control, however one concern with this method is the risk of cell death when a target protein is vital to cell survival. Other initiatives dispense with negative controls and verify positive identification instead. Mass spectrometry, for example, yields unique spectra that differentiate an antibody-bound protein of interest from any other bound molecule but is limited only to those proteins that can be immunoprecipitated.

While all of these approaches sound simple, their methodologies can be complex. Anyone seeking a deeper understanding of siRNA knockdown validation studies, or considering their own validation protocol, may find the following overview useful. These steps are based on Proteintech methods, which have been tested and refined over years. However, they are by no means the only way to perform this validation.

Design and engineer a vector

Selecting a target and designing an appropriate vector to transfect into cells is a relatively straightforward process, with an abundance of literature and online resources available to guide the process (RNAi Consortium,    Dharmacon,    Ui-Tei, and   Genelink). The actual engineering, of course, requires a bit more hands-on finesse to create the short hairpin RNA that is the precursor form of the siRNA. The ultimate aim is to design two single-stranded 19-22mer DNA oligonucleotides (one sense strand, one antisense strand) whose transcription products will eventually anneal together, linked at one end by a short loop sequence. Proteintech uses the loop sequence TTCAAGACG.

Transfect and culture

Once the vector has been generated and produced in sufficient quantity, scientists need to determine an appropriate transfection method for delivery into cells. Upon successful transfection, the cells transcribe the foreign DNA to generate the shRNA described above (Figure 2). Afterward, Dicer processes the shRNA into siRNA by removing the loop sequence. The resulting siRNA binds with RISC (RNA-induced silencing complex), which separates the two strands of the RNA and activates the complex. RISC remains bound to one strand, that complementarily binds to a target mRNA and cleaves it. This suppresses production of the associated protein. Scientists need to allow time for the biological processes to be carried out, while culturing the cells to produce enough sample for a Western blot.

Figure 2. An overview of the biological pathway of siRNA knockdown.

Cell death can be a major source of frustration, particularly if the target protein is vital to cell survival. Fortunately, most cells can propagate normally if the protein is suppressed, but not completely eliminated. In such cases, knockdown methods can be fine tuned and are likely a preferred alternative to knock out validation, which prevents any transcription through gene editing.

Test and evaluate

A strong signal for the empty-vector cells next to a weak signal for the siRNA-transfected cells in a Western blot means that the antibody is specific and that the knockdown experiment was successful. Any nonspecific bands should raise questions, as these could indicate that the antibodies themselves are nonspecific. Bands should also be consistent across all lanes of the Western blot membrane. If not, something may have gone wrong in the experiment, and protocol and experimental design should be reviewed.

While the value of negative controls to antibody validation protocols should not be understated, it is also understood that such validation is not widely available and that it consumes time and resources to perform in individual labs. Thorough Western blot validation with only positive controls can be a second-best alternative, provided that there is a sufficient quantity of data, and corroborated by published studies that reference the reagent catalog number and manufacturer. We encourage the development and use of negative controls such as siRNA knockdown as the new standard in antibody validation. Doing so will elevate reproducible research and accelerate scientific progress.

References

Genetic knockdown and knockout are complementary tools for assessing antibody specificity. Before building constructs and ordering reagents, consider which one is best for your situation. Each method has its advantages and limitations: 

Method 

Best for 

Limitations and considerations

Knockdown 

  • Essential genes 

  • Genes with pre-designed RNAi reagents  

  • Efficiency can be an issue 

  • Off-target effects 

  • Residual signal may still reflect non-specific binding 

  • May not see effect on very highly expressed proteins 

  • If protein turnover is low, effect may be hard to see 

  • Usually transient effect 

Knockout 

  • High expression genes 

  • High precision necessary 

  • Some genes cannot be targeted  by CRISPR, TALENs, etc. 

  • Essential genes cannot be knocked out 

標的サイトの選択

利用可能な文献やオンラインのshRNA設計ツールを確認し標的サイトを決定します。

pGenesil-1-shRNA発現ベクターの構築

標的shRNAをコードするセンス鎖と、相補鎖(アンチセンス鎖)の、2種類の一本鎖DNAオリゴヌクレオチド(19~22mer)を設計します。2つを細胞内で発現させると、両者がアニーリングして最終的に二本鎖オリゴヌクレオチドを生成します。

ループ配列

2つの配列(センス鎖とアンチセンス鎖)は、発現時にヘアピンループ構造を形成する短いリンカー配列によって同じ鎖上で連結しています。様々なループ配列が機能的に利用できることが報告されていますが、プロテインテックではTTCAAGACGの配列を使用しています。

その他の詳細

shRNA鋳型の末端には、終結シグナルとして6ヌクレオチドのポリ(T)テールが付加されています。2種類のオリゴヌクレオチド鎖の5'末端は非相補的で、制限部位BamHⅠとHindⅢで突出末端を形成しpGenesil-1ベクターへの方向性クローニングを効率的に行うことができます。

RNAi(RNA干渉:RNA interference)は、配列特異的な遺伝子サイレンシングを実験室レベルで実施できる比較的新しい技術です。RNAiは、当初植物で発見され、後に動物でも確認された同名の生物学的プロセスに基づいています。自然界では、RNAiは寄生ヌクレオチド配列(例:ウイルスやトランスポゾン)から細胞を防御する際に重要な役割を担っており、発生にも影響します。

RNAiは、遺伝子転写からタンパク質翻訳の中間段階であるRNAレベルでの遺伝子発現制御機構であり、タンパク質生合成のためのメッセンジャーRNA(mRNA)を阻害することにより機能します。研究室の実験では、細胞等への合成dsRNAの導入やshRNA(small hairpin RNA)をコードするプラスミドDNAベクターの導入によって行われます。dsRNAやshRNAはその後プロセシングを受け一本鎖siRNAとなり、このsiRNAは相補的配列をもつmRNAと結合します。siRNAの結合により標的mRNAが「サイレンシング」されると、細胞内プロセスを経て分解されます。RNAi法は、遺伝子の「ノックダウン」の一形態で、成功すると標的タンパク質の発現レベルがウェスタンブロットの検出下限以下に著しく低下します。その際、ウェスタンブロット膜上でshRNA処理サンプルのシグナルが消失(または著しく減少)している場合、抗体が特異的であることを同定できます。

図1. プラスミド発現ベクターによりsiRNAを細胞で発現させた際のRNAiの作用(概要)