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Laboratory Chemicals > microRNA Catalog


microRNA Catalog ( published in April, 2014)


Contents

  1. microRNA and Ago-Subfamily Proteins
  2. microRNA Experiment Flow
  3. Summary of Ago Immunoprecipitation
  4. Anti-Ago Monoclonal Antibody Series
  5. microRNA Isolation Kit Series
  6. MagCapture™ microRNA Isolation Kit(magnetic beads)
  7. microRNA Isolation Kit(silica-based beads)
  8. Total RNA Purification
  9. microRNA Analysis Tools
  10. cDNA Cloning
  11. Microarray Analysis
  12. Wako Pure Chemical Industries, Ltd. Publication list
  13. Products List


microRNA Catalog published in 2014




microRNA and Ago-Subfamily Proteins

microRNA (miRNA) are small functional RNA molecules (about 22 nucleotides) which act as posttranscriptional regulators of gene expression. They serve many functions within biological organisms (Ref. 1), and more than 1,000 types are known to exist in humans and mice. Worldwide there are many efforts to identify new types of microRNA with unknown functions and elucidate functions of those involved in disease. Recently it has been reported that microRNA exist not only within cells, but also blood and other bodily fluids. They are now garnering attention as possible clinical markers for cancer and other diseases (Ref. 2).

In cells, microRNA pass through many steps before being incorporated into a multiprotein complex called a RISC (RNA-induced silencing complex). After binding with the main component of Agosubfamily proteins, they bind with mRNA targets and either cleave them or inhibit translation, thus regulating gene expression (Fig. 1) (Ref. 3, 4, 5). The Ago-subfamily of proteins are characterized by containing both a PAZ domain and a PIWI domain (Fig. 2). There are four types in humans (hAgo1 to hAgo4), and although each expression levels differ from each them in each cell lines (Fig. 3) (Ref. 6). The most commonly expressed Ago-subfamily protein is Ago2, which has unique "Slicer" activity which directly cleaves target RNA. Thus, Ago2 is considered to play an important role in microRNA pathways (Ref. 7, 8, 9, 10, 11, 12, 13). Anti-Ago antibodies are used for immunoprecipitation of RISC, allowing recovery of both microRNA and their mRNA targets. This immunoprecipitation method is vital for elucidating the various functions of microRNA (Ref.14, 15, 16, 17, 18, 19, 20, 21).

We offer a full selection of antibodies for various human and mouse Ago-subfamily proteins, as well as immunoprecipitation kits for them. These tools allow comprehensive analysis of microRNA and mRNA contained in RISC in cells and tissue, as well as Ago-bound microRNA in blood. Fig.1 Molecular mechanism of RNA silencing
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References
  • 1.Bartel DP: MicroRNA:genomics, biogenesis, mechanism, and function. Cell 2004, 116:281-297.
  • 2. Cortez MA, Calin GA : MicroRNA identification in plasma and serum: a new tool to diagnose and monitor diseases.
    Expert Opin Biol Ther
    2009, 9:703-711
  • 3. Hammond SM, Bernstein E, Beach D, Hannon GJ: An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells.
      Nature 2000, 404:293-296.
  • 4. Hammond SM, Boettcher S, Caudy AA, Kobayashi R, Hannon GJ: Argonaute2, a link between genetic and biochemical analyses of RNAi.
      Science 2001, 293:1146-1150.
  • 5. Gregory RI, Chendrimada TP, Cooch N, Shiekhattar R: Human RISC couples microRNA biogenesis and posttranscriptional gene silencing.
      Cell 2005, 123:631-640.
  • 6. Sasaki T, Shiohama A, Minoshima S, Shimizu N: Identification of eight members of the Argonaute family in the human genome.
      Genomics 2003, 82:323-330.
  • 7. Pillai RS, Artus CG., Filipowicz W: Tethering of human Ago proteins to mRNA mimics the miRNA-mediated repression of protein synthesis.
      RNA 2004, 10:1518-1525.
  • 8. Meister G, Landthaler M, Patkaniowska A, Dorsett Y, Teng G, Tuschl T: Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs.
    Mol Cell 2004, 15:185-197.
  • 9. Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ, Hammond SM, Joshua-Tor L, Hannon GJ: Argonaute2 is the catalytic engine of mammalian RNAi.
      Science 2004, 305:1437-1441.
  • 10. Song JJ, Smith SK, Hannon GJ, Joshua-Tor L: Crystal structure of Argonaute and its implications for RISC slicer activity.
      Science 2004, 305:1434-1437.
  • 11. Meister G, Tuschl T: Mechanisms of gene silencing by double-stranded RNA.
      Nature 2004, 431:343-349.
  • 12. Hutvagner G, Zamore PD: A microRNA in a multiple-turnover RNAi enzyme complex.
      Science 2002, 297:2056-2060.
  • 13.Yekta S, Shih IH, Bartel DP: MicroRNA-directed cleavage of HOXB8 mRNA.
      Science 2004, 304:594-596.
  • 14. Beitzinger M, Peters L, Zhu JY, Kremmer E, Meister G: Identification of human microRNA targets from isolated Argonaute protein complexes.
    RNA Biol 2007, 4:76-84.
  • 15. Karginov FV, Conaco C, Xuan Z, Schmidt BH, Parker JS, Mandel G, Hannon GJ: A biochemical approach to identifying microRNA targets.
      Proc Natl Acad Sci USA 2007, 104:19291-19296.
  • 16. Easow G, Teleman AA, Cohen SM: Isolation of microRNA targets by miRNP immunopurification.
      RNA 2007, 13:1198-1204.
  • 17. Hendrickson DG, Hogan DJ, Herschlag D, Ferrell JE, Brown PO: Systematic identification of mRNAs recruited to Argonaute2 by specific microRNAs and corresponding changes in transcript abundance.
     PloS ONE 2008, 3:e2126.
  • 18. Landthaler M, Gaidatzis D, Rothballer A, Chen PY, Soll SJ, Dinic L, Ojo T, Hafner M, Zavolan M, Tuschl T: Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs.
      RNA 2008, 14:2580-2596.
  • 19. Chi SW, Zang JB, Mele A, Darnell RB: Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps.
      Nature 2009, 460:479-486.
  • 20. Wang WX, Wilfred BR, Hu Y, Stromberg AJ, Nelson PT: Anti-Argonaute RIP-Chip shows that miRNA transfections alter global patterns of mRNA recrui™ent to microribonucleoprotein complexes.
      RNA 2009 Dec.30.
  • 21. Hayashida Y, Nishibu T, Inoue K, Kurokawa T: A useful approach to total analysis of RISC associated RNA. BMC Res Notes 2009, 2:169.
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1. microRNA Experiment Flow

We offer a microRNA Isolation Kit series containing monoclonal antibodies for various Ago-subfamily proteins and immunoprecipitation methods (Ago IP) for each of these. We also offer tools such as the ISOGEN® series for Purification of total RNA from samples or the microRNA Extractor ® SP Kit, all allowing purification of fractions containing microRNA from samples depending on research needs. Finally, we offer additional powerful tools to support microRNA research, such as tools to clone cDNA from purifi ed microRNA or target mRNA, electrophoresis tools, and microarray analysis services.

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2. Summary of Ago Immunoprecipitation

We offer a full selection of human and mouse antibodies for Ago-subfamily proteins as well as the microRNA Isolation Kit series based on immunoprecipitation (Ago IP) using these antibodies. These tools offer the following benefi ts over traditional total RNA purification.



Benefit 1 Concentration of Ago-bound microRNA

Improvement of detection sensitivity of microRNA incorporated in RISC and suppress detection of non-specifi c detection of microRNA not incorporated in RISC.

Example:Comparing populations of Ago IP RNA fractions and total RNA fractions from HeLa cells

Starting with HeLa cells, we used a micro-array (3D-Gene®, Toray) to compare populations of RNA obtained with immunoprecipitation from monoclonal antibodies specifically recognizing each Ago subfamily protein (Ago1 to Ago4) (Ago IP RNA fraction) with microRNA contained in a total RNA fraction prepared using ISOGEN (AGPC) (Fig. 1). Results showed that more microRNA was concentrated in each Ago IP RNA fraction than the total RNA fraction (excluding the Ago4 IP RNA fraction), yielding a stronger signal (Fig. 2). One signal was strongly detected in the total RNA fraction while remaining largely absent in the Ago IP RNA fraction, suggesting that microRNA not incorporated into RISCs were non-specifi cally detected in the total RNA fraction.








Benetif 2: Concentration of RISC-binding target mRNA

RNA fractions obtained by Ago IP method are applicable to analysis of target mRNA by cDNA cloning, qPCR, microarray, Next-generation DNA sequencer because target mRNA is concentrated.

Example:Concentration of target mRNA of liver-specific microRNA(miR-122)

Either miR-122 or the control of luciferace siRNA (Luc siRNA) were delivered to HepG2, a liver cancer cell line with low expression of miR-122, then Ago2 IP RNA (using anti-Ago2 monoclonal antibody 4G8) and total RNA (using ISOGEN) purified from both samples. Quantitative PCR was then used to compare (Fig. 1) quantities of Aldo A mRNA as a miR-122 target (normalized with GAPDH mRNA) in both cell types. Results show that the quantity of Aldo A mRNA decreased by RISC cleavage, in the total RNA fraction while Aldo A mRNA in the Ago2 IP RNA fraction was concentrated (Fig. 2).



Benefit 3: Concentration of free Ago-bound microRNA from blood samples

Blood is known to include microRNA contained in exosomes, HDL-bound microRNA, and Ago-bound microRNA. Ago immunoprecipitation method allows for specific isolation of Ago-bound microRNA, and it can be used for biomarker searches and other uses.


Example 1:Isolation of Ago2-bound microRNA from human plasma

Quantitative PCR (Ct value) was used to measure 12 types of arbitrarily-selected microRNA in pooled plasma of healthy individuals from immunoprecipitated Ago2 IP RNA (using anti-Ago2 monoclonal antibody 4G8) and total RNA (using microRNA Extractor® SP Kit) (Fig. 1). Comparison reveals that most microRNA detected in the total RNA sample was also detected in the Ago2 IP RNA fraction, while types such as miR-22 and miR-92a were highly concentrated in the Ago2 IP RNA fraction (low Ct value compared to total RNA) (Fig. 2).

Fig.2 Comparison of microRNA quantities


Example2:Isolation of each Ago-bound microRNA from human plasma

Quantitative PCR was used to compare microRNA (miR-92a and miR-122) contained in immunoprecipitated RNA (Ago 1, 2, 3, 4 IP RNA) using anti-Ago (Ago1 to Ago4) monoclonal antibodies from pooled plasma of healthy individuals (Fig. 1). Results show microRNA present in other Ago IP RNA fractions in addition to the Ago2 IP RNA fraction, with liver-specific miR-122 highly concentrated in the Ago3 IP RNA fraction (Fig. 2).




Fig.2 Comparison of microRNA quantities from each Ago IP RNA fractions
Fig.1 Experiment flow
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3. Anti-Ago Monoclonal Antibody Series

We offer a full line of mouse monoclonal antibodies both for Western blot detection and immunoprecipitation recognizing various human and mouse Ago-subfamily proteins. These tools allow selective immunoprecipitation of various Ago-subfamily proteins from human and mouse samples in order to obtain microRNA bonded to each. Proteins obtained can also be confi rmed with Western blot.

Antigen Cross-reactivity Description Clone No. Subclass Application Concentration
Ago1 Human, Mouse Anti Ago1, Monoclonal antibody 2A7 IgG2a・k IP, ICC, RIP 1.0mg/mL
Anti Ago1, Monoclonal antibody 1F2 IgG2a・k WB
Ago2 Human Anti Human Ago2, Monoclonal antibody 4G8 IgG1 IP, WB
ICC, RIP
Mouse Anti Mouse Ago2, Monoclonal antibody 2D4 IgG1 IP, WB
ICC, RIP
Ago3 Human Anti Human Ago3, Monoclonal antibody 1C12 IgG1 IP, RIP
Anti Ago3, Monoclonal antibody 6-107 IgG1・k WB
Mouse Anti Mouse Ago3, Monoclonal antibody S09 IgG1・k IP, RIP
Anti Mouse Ago3, Monoclonal antibody S011 IgG1・k WB
Ago4 Human, Mouse Anti Ago4, Monoclonal antibody 2G7 IgG2b IP, RIP
Anti Ago4, Monoclonal antibody 2B2 IgG1 WB
Ago1-4 Human, Mouse Anti Ago1-4, Monoclonal antibody 1G3 IgG1 IP, WB



Immunoprecipitation of recombinant Ago proteins

We evaluated immunoprecipitation performance of each antibody using human and mouse recombinant Ago proteins (DYKDDDDK tag was fused to C-terminal). Each anti-Ago1 to anti-Ago4 antibody specifically immunoprecipitated each recombinant Ago protein, demonstrating that anti-Ago1 to anti-Ago4 antibodies are capable of immunoprecipitation of all Ago-subfamily proteins.

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Western blot analysis of recombinant Ago proteins

We evaluated Western blot performance of each antibody using human and mouse recombinant Ago proteins (DYKDDDDK tag was fused to C-terminal). Each anti-Ago1 to anti-Ago4 antibody specifically detected each recombinant Ago protein, demonstrating that anti- Ago1 to anti-Ago4 antibodies are capable of detection of all Ago-subfamily proteins.

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Endogenous Ago protein immunoprecipitation and Western blot analysis

We immunoprecipitated and then used Western blot analysis for detection of endogenous Ago proteins using anti-human Ago antibodies from human K562 cells and anti-mouse Ago antibodies from mouse P388D1 cells or mouse lung tissue. Each anti-Ago1 to anti-Ago4 antibody specifically immunoprecipitated each endogenous Ago protein and used for Western blot analysis, indicating that anti-Ago1 to anti- Ago4 antibodies are capable of immunoprecipitation of endogenous Ago1 to Ago3.

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4. microRNA Isolation Kit Series

We offer a microRNA Isolation Kit series to simplify an entire range of procedures from Ago immunoprecipitation to RNA Purification. This kit series allows recovery of Ago-bound microRNA and target mRNA from both human and mouse cells and tissues, and free Ago-bound microRNA from culture supernatant, serum, and plasma. Obtained RNA can then be analyzed by quantitative PCR, microarrays, and next-generation sequencing. This series includes MagCapture™ microRNA Isolation Kits, which adopt magnetic beads, and microRNA Isolation Kits, which adopt silica-based beads. Features and benefi ts of each product are described below.

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4-1. MagCapture™ microRNA Isolation Kit(magnetic beads)

We offer four types of MagCapture™ microRNA Isolation Kits: three types contain magnetic beads immobilized to anti human Ago2 antibody (4G8), anti mouse Ago2 antibody (2D4), or anti-Ago1-4 antibody (1G3), and one type contain with Protein G magnetic beads which can immobilize an optional anti Ago subfamily antibodies. All of these kits adopt magnetic beads for easy immunoprecipitation. After immunoprecipitation, two protocols for the RNA Purification step are selectable: One-step method for obtaining high-yield microRNAs by a simple procedure or Spin column method for obtaining highpurity microRNAs.



Kit contents(Example: Human Ago2, 10 reactions)

  1. Anti-Human Ago2 Magnetic Beads Solution・・600μL×1tube
  2. Cell Lysis Solution・・・・・・・・・・・・・・20mL×1tube
  3. Washing Solution for IP・・・・・・・・・・・ 40mL×1tube
  4. Elution Solution I for IP・・・・・・・・・・・ 500μL×1tube
  5. Elution Solution II for IP・・・・・・・・・・・500μL×1tube
  6. Binding Solution for Spin Column・・・・・・・2mL×1tube
  7. Binding Enhancer for Spin Column・・・・・・100μL×1tube
  8. Washing Solution I for Spin Column・・・・・3mL×1tube
  9. Washing Solution II for Spin Column・・・・ 4mL×1tube
  10. Elution Solution for Spin Column・・・・・・ 1mL×1tube
  11. Spin Column/Collection Tube・・・・・・・・10tubes

Outline


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Description Antibody
clone no.
Animal Compatible samples RecoveredRNA IP B/F separation method RNA purification method
MagCapture™ microRNA Isolation Kit, Human Ago2 4G8 Human, monkey, dog Cell, tissue,
serum, plasma
microRNA and
target mRNA
Magnetic separation Spin column or
one-step
MagCapture™ microRNA Isolation Kit, Mouse Ago2 2D4 Mouse, rat, dog
MagCapture™ microRNA Isolation Kit, Ago1-4 1G3 Human, monkey, dog,
mouse, rat
microRNA
MagCapture™ microRNA Isolation Kit, Protein G Any Depends on antibody Cell, tissue Depends on antibody Spin column


MagCapture™ microRNA Isolation Kit, Human Ago2

Recovering microRNA from human cell strains

We used MagCapture™ microRNA Isolation Kit, Human Ago2 and a previous product to obtain microRNA from human K562 cells(1×107 cellscells), then compared quantities of microRNA obtained with denaturing polyacrylamide gel electrophoresis (silver staining) (Fig. 1) and quantitative PCR (miR-92a quantity) (Fig. 2). Results showed that the two products yielded equivalent quantities of microRNA.

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Recovering free Ago2-bound microRNA from human plasma

We used MagCapture™ microRNA Isolation Kit, Human Ago2 and a previous product to obtain microRNA from pooled plasma of healthy individuals, then compared quantities obtained with quantitative PCR (miR-92a quantity). As shown below, using the spin column method with the MagCapture™ resulted in approximately equivalent yield to our previous product, while the one-step method demonstrated greater quantities of microRNA recovered compared to our previous product.

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Recovering target mRNA of microRNA

Either miR-122 or the control of luciferase siRNA (Luc siRNA) were delivered to HepG2, a liver cancer cell line with low expression of miR-122, then immunoprecipitated Ago2 IP RNA obtained using either MagCapture™ microRNA Isolation Kit, Human Ago2 or our previous product. Total RNA was prepared with ISOGEN. Quantitative PCR was then used to compare quantities of miR-122 (Fig. 4) and its target mRNA (Aldo A corrected with GAPDH mRNA) (Fig. 5). Results showed that miR-122 and Aldo A mRNA were concentrated in both MagCapture™ and our previous kit, while the former also allowed recovery of microRNA's target mRNA. In contrast, total RNA obtained with ISOGEN showed decreased quantities of Aldo A mRNA due to cleavage accompanying increase of miR-122.

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MagCapture™microRNA Isolation Kit, Mouse Ago2

Recovering microRNA from mouse cell strains

We used MagCapture™ microRNA Isolation Kit, Human Ago2 and a previous product to obtain microRNA from mouse P388D1 cells(2×107 cells), then compared quantities of microRNA obtained with denaturing polyacrylamide gel electrophoresis (silver staining) (Fig. 1) and quantitative PCR (miR-92a quantity) (Fig. 2). Results showed that the two products yielded equivalent quantities of microRNA.

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Recovering free Ago2-bound microRNA from rat plasma

We used MagCapture™ microRNA Isolation Kit, Mouse Ago2 and a previous product to obtain microRNA from 200 μL rat plasma, then compared quantities obtained with Quantitative PCR (miR- 92a quantity). As shown below, using the spin column method with the MagCapture™ resulted in approximately equivalent yield to our previous product, while the one-step method demonstrated greater quantities of microRNA recovered compared to our previous product.

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MagCapture™ microRNA Isolation Kit, Ago1-4

Recovering Ago1-4-bound microRNA

We used a microarray(3D-Gene®,Toray) to compare microRNA expression profile of RNA fractions obtained from HeLa cells by immunoprecipitation of anti-Ago1 to anti-Ago4 antibodies. While large quantities of miR-22 and miR-16 were incorporated into Ago2, large quantities of miR-1260b and miR- 4286 were incorporated into Ago1, Ago3, and Ago4 (see table below).
Based on these results, we decided to study whether MagCapture™ could recover microRNA binding Ago1, Ago3, or Ago4 at high levels, which was diffi cult with our previous product (microRNA Isolation Kit, Human Ago2 ). Both products were used to recover microRNA from HeLa cells (1×107 cells), and quantitative PCR used to measure quantities of miR-22, miR-16, miR-1260b, and miR-4286 recovered, using miR-92a as an internal standard. Results showed equivalent recovery between the two products of miR-22 and miR-16, which are shown present in Ago2 in high levels, while MagCapture™ microRNA Isolation Kit, Ago1-4 recovered miR-1260b and miR-4286 at higher levels than the previous product (see below). These results show that the new MagCapture™ microRNA Isolation Kit, Ago1-4 is capable of the detection of not only microRNA binding Ago2, but also in Ago1, Ago3, and Ago4.

  Signal intensity Signal ratio
Name Ago1 IP Ago2 IP Ago3 IP Ago4 IP Ago2/Ago1 Ago2/Ago3 Ago2/Ago4
hsa-miR-22 3521.4 21696.5 3771.2 292.4 6.16 5.75 74.20
hsa-miR-16 6886.3 28527.2 5013.4 582.5 4.14 5.69 48.97
hsa-miR-1260b 2060.3 540.9 1270.6 1977.9 0.26 0.43 0.27
hsa-miR-4286 678.8 202.4 2019.2 471.2 0.30 0.10 0.43

Table 1 Result of microarray analysis

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Recovering microRNA from human and mouse cell lines

MagCapture™ microRNA Isolation Kit, Ago1-4 and our previous product (microRNA Isolation Kit, Human Ago2 or Mouse Ago2) were used to recover microRNA from human K562 cells(1×107 cells) and mouse P388D1 cells(2×107 cells). Quantitative PCR (miR-92a quantity) comparisons shown below indicate that MagCapture™ recovered more microRNA more than the previous product.

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Recovering free Ago-binding microRNA from human and rat plasma

MagCapture™ microRNA Isolation Kit, Ago1-4 and our previous product (microRNA Isolation Kit, Human Ago2 or Mouse Ago2) were used to recover microRNA from 200 μL human and rat plasma. Quantitative PCR (miR-92a quantity) comparisons shown below indicate that MagCapture™ recovered microRNA more than the previous product.

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Recovering microRNA from various animal cell lines

MagCapture™ microRNA Isolation Kit, Human Ago2, Mouse Ago2, and Ago1-4 were used to immunoprecipitate microRNA from K562 cells, monkey COS-7 cells, dog MDCK cells, mouse P388D1 cells, and rat SCC-131 cells (each: 2×106 cells). On the other hand, ISOGEN was used to obtain total microRNA fractions from the same cell samples.
As a result of comparison of relative value of miR-92a quantities between each Ago IP fractions and total RNA fractions, MagCapture™ recovered microRNAs from all cell types.

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MagCapture™ microRNA Isolation Kit, Protein G

Recovering microRNA from human and mouse cell lines (product comparison)
MagCapture™ microRNA Isolation Kit, ProteinG (using anti-Ago1 (2A7), anti-Ago2 (4G8), and anti- Ago3 (1C12) antibodies) and our previous products were used to obtain microRNA from human K562 cells (1×107 cells). Figure 1, 2, 3 shows quantitative PCR (miR-92a quantity) comparisons of quantities of microRNA recovered. Next, MagCapture™ (using mouse anti- Ago2 antibodies (2D4) and our previous product were used to obtain microRNA from mouse P388D1 cells (2×107 cells) (Fig. 4). Results show that MagCapture™ with an optional anti-Ago antibody enables microRNA recovery superior to that of our previous product.







Recovering different types of Agobinding microRNA from human and mouse cell lines
MagCapture™ microRNA Isolation Kit, ProteinG (using anti-Ago1 (2A7), anti-Ago2 (4G8), anti-Ago3 (1C12), and anti-Ago4 (2G7) antibodies) were used to obtain microRNA from human K562 cells (1×107 cells). Quantities of microRNA were measured with denaturing polyacrylamide gel electrophoresis (silver staining) and quantitative PCR (miR-92a quantity) (Fig. 5). Next, MagCapture™ (using anti-Ago1 (2A7), anti-Ago2 (2D4), anti-Ago3 (S09), and anti-Ago4 (2G7) antibodies) was used to obtain microRNA from mouse P388D1 cells (2×107 cells). Quantities of microRNA were measured with denaturing polyacrylamide gel electrophoresis (silver staining) and quantitative PCR (miR-92a quantity) (Fig. 6). Results show that MagCapture™ with an optional anti-Ago antibody enables recovery of each Ago-binding microRNA.

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microRNA Isolation Kit(silica-based beads)

Our microRNA Isolation Kit uses anti-Ago antibodies conjugated to silica-based polymer beads with low non-specific absorbency for centrifuge immunoprecipitation. Post-immunoprecipitation RNA purification steps are performed by phenol/ chloroform and ethanol precipitation. We offer a lineup of four types: anti-Ago1 antibody (2A7), anti human Ago2 antibody (4G8), anti mouse Ago2 antibody (2D4), and anti human Ago3 antibody (1C12).



Kit contents (Example: Human Ago2, 10 reactions)

  1. Anti-Human Ago2 Antibody Beads Solution
     500µL×1tube
  2. Cell Lysis Solution
     50mL×1tube
  3. Elution Solution
     500µL×1tube
  4. Ethachinmate
     30µL×1tube
  5. 3 mol/L Sodium Acetate
     400µL×1tube

Outline


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Description Antibody
clone no.
Animal Compatible samples Recovered RNA IP B/F separation method RNA purification method
microRNA Isolation Kit, Human/Mouse Ago1 2A7 Human, mouse Cells, tissue microRNA and
target mRNA
Centrifuge Phenol/
chloroform
microRNA Isolation Kit, Human Ago2 4G8 Human, monkey, dog
microRNA Isolation Kit, Mouse Ago2 2D4 Mouse, rat
microRNA Isolation Kit, Human Ago3 1C12 Human

Application Data

The microRNA Isolation Kit, Human Ago2 was used to purify microRNAs from human HeLa, HepG2, and HEK293 cell lines (5 × 106 cells) and mouse P388D1 cell lines (5 × 106 cells). The purifi ed microRNAs were detected by silver staining (311-03961, CLEAR STAIN Ag, 20stains) after Urea-PAGE. As a result, the microRNA Isolation Kit, Human Ago2 purifi ed microRNAs specifi cally from human HeLa, HepG2, and HEK293 cell lines. In addition, the microRNA Isolation Kit, Mouse Ago2 was used to purify microRNAs from human HeLa cell lines (5 × 106 cells) and rodent P388D1, CHO, PC-12 cell lines (5 × 106 cells). The purifi ed microRNAs were detected by the previous described method. As a result, the microRNA Isolation Kit, Mouse Ago2 purifi ed microRNAs specifi cally from rodent P388D1, CHO, PC-12 cell lines.

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Total RNA Purification

microRNA Extractor® SP Kit

This kit is for extraction of microRNA from serum or plasma of humans and animals. It enables microRNA extraction without use of phenol, chloroform, and other hazardous reagents required in previous methods.

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Code No. Description Package size
295-71701 refrigeratormicroRNA Extractor® SP Kit 50 Extractions


ISOGEN®II

ISOGEN®IIis a reagent for extracting total RNA or small RNA from animal tissue or cultured cells. It is a uniform fluid containing phenol and guanidine that can isolate RNA in a single step through interactions with cellular components. There is not require to separate the liquid phase with chloroform as previous products (ISOGEN®or ISOGEN®-LS).


image Total RNA was extracted with ISOGEN® and ISOGENR II, then analyzed with a 3D-Gene® Human microRNA Oligo chip (Toray). Results show ISOGEN® II efficacy in small RNA extraction. image


Code No. Description Package size
317-07363 refrigeratorISOGEN® II 10ml
311-07361 100ml


ISOGEN® ISOGEN®-LS

ISOGEN® and ISOGEN®-LS enable the isolation of undamaged RNA in high yield about 1 hour without contaminating DNA or protein. Therefore, Northern analysis or dot blot hybridization can be performed without DNase or other processing (Fig. 1).

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Sample Yield
Tissue Mouse Liver
Spleen
Kidney
Skeletal
Brain
Placenta
6~10 µg RNA/mg tissue
6~10 µg RNA/mg tissue
3~4 µg RNA/mg tissue
1~1.5 µg RNA/mg tissue
1~1.5 µg RNA/mg tissue
1~4 µg RNA/mg tissue
Rat, pituitary gland, or human liver
needle biopsy sample
4~8 µg RNA/mg tissue
Plant Arabidopsis or tobacco 150~200 µg RNA/0.5~1 g weight tissue
Cultured cells Epithelial
Fibroblasts
8~15 µg RNA/106 cell
5~7 µg RNA/106 cell
Blood Human or animal 7~15 µg RNA/mL blood


Code No. Description Package size
315-02504 refrigeratorISOGEN® 10ml
317-02503 50ml
311-02501 100ml


Code No. Description Package size
317-02623 refrigeratorISOGEN®-LS 10ml
311-02621 100ml
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microRNA Analysis Tools

Electrophoresis

image This product is an polyacrylamide gel with 4M urea. It is a ready-touse precast gel, so it does not require steps such as dissolving urea or preparing gels. Acrylamide concentration is 15%.

image

Code No. Description Package size
194-15881 refrigeratorSuperSep™ RNA, 15%, 17well 5 sheets


R-Mark™ small RNA Marker(20-50nt)

This product is consisted of four types of single-chain RNA (20, 30, 40, 50 nt). Use after mixing with RNA loading buffer.

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  • Recommended quantity: 1 μL/lane (50 times)
  • Component: Water (RNase free)
  • Total RNA quantity: 112.5µg/mL
  • RNA quantity in each band/µL
  • 20nt(37.5ng),30nt,40nt,50nt(25 ng each)

Code No. Description Package size
182-02711 freezeR-Mark™ small RNA Marker 50μl


CLEAR STAIN Ag

CLEAR STAIN Ag is a silver staining kit developed especially for nucleic acids (DNA/RNA), enabling high sensitivity detection after polyacrylamide gel electrophoresis with low background noise. This method offers 50 to 100 higher sensitivity than ethidium bromide staining (measured at 302 nm), allowing detection of 20 to 50 pg DNA per band.

Kit contents (140×140×1mm 6% polyacrylamide gel, 20 stains)

  1. Fixing solution (×20): CTAB, ethanol, 200 ml
  2. Ammonia solution (×20): ammonia, 200 ml
  3. Staining solution A (×20): silver nitrate, 200 ml
  4. Staining solution A (×20): sodium hydroxide, ammonia, 200 ml
  5. Developing solution A(×10): sodium carbonate, 200 ml×2 (10× concentrate)
  6. Developing solution B(×20): formaldehyde, sodium thiosulfate, 200 ml
  7. Preserving solution (×20): glycerol, acetic acid, 200 ml
  8. Product manual

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  • Sample: Marker 9 (φX174/Hin f I digest)
  • Sample quantity: From left: 500, 250, 125, 63, 32, 16, 8, 4,
    2, 1, 0.5, 0.25ng
  • Buffer: TBE buffer
  • Gel size: 140×140×1 mm (6% polyacrylamide gel)


Code No. Description Package size
311-03961 Clear Stain Ag 20 stains


5×TBE

  • Components
  • 0.445 mol/L Tris-borate, 10 mmol/L EDTA

  • Storage
  • Room temp.

  • Comments
  • Dilution amounts
    Agarose gel electrophoresis: About 10×
    Polyacrylamide gel electrophoresis: About 5×



Code No. Description Package size
318-90041 5×TBE 1,000ml

Usage Example of Electrophoresis Products

MagCapture™ microRNA Isolation Kit, Human Ago1-4 was used to obtain an RNA fraction from human K562 cells (1×107 cells), then denaturing polyacrylamide gel electrophoresis performed with SuperSep RNA, 15%, 17-well, with CLEAR STAIN Ag used for silver staining detection. Result show the detection of microRNA of about 22 bp in the RNA fraction.

image Lane 1: R-Mark™ Small RNA Marker 1μL 1/50 diluted fluid
Lane 2: Control siRNA duplex, Firefl y Luciferases GL3(22mer) 0.5ng
Lane 3: MagCaputure™ microRNA Isolation Kit, Ago1-4 Spin column method
Lane 4: MagCaputure™ microRNA Isolation Kit, Ago1-4 One-step method


Gel: SuperSep™ RNA, 15%, 17well
Running buffer: 0.5×TBE
Current: 10 mA constant for 75 min.
Stain: Clear Stain Ag (staining time: 60 min., developing time: 5 min.)

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cDNA Cloning

microRNA Cloning Kit Wako

microRNA Cloning Kit Wako adopt our original buffer solution which allows two reactions in the same solution: dephosphorylation by shrimp alkaline phosphatase (SAP), which is easily inactivated with heat treatment; and efficient adaptor ligation of single-strand DNA/RNA with a thermostable ligase (sold separately). This allows simple and efficient adaptor ligation reactions, enabling easy cDNA synthesis from microRNA.



Features

  1. Highly-efficient and accurate adaptor ligation by thermostable ligase.
  2. Suitable for cloning of microRNA forming secondary structures.
  3. Reduced RNA operations means cDNA coding microRNA can be synthesized within 1.5 days.

Kit contents (8 reactions)

  1. SAP・・・・・・・・・・・・・・・16μl
  2. 5×SAP Buffer・・・・・・・・・・ 64μl
  3. 40×Ligation Buffer・・・・・・・ 16μl
  4. RNase Inhibitor・・・・・・・・・16μl
  5. 10mmol/L MnCl2・・・・・・・・ 16μl
  6. Reverse Transcriptase・・・・・・ 8μl
  7. 10×RT Buffer・・・・・・・・・・16μl
  8. dNTP Mixture・・・・・・・・・ 112μl
  9. 0.5 mol/L EDTA, pH8.0・・・・・ 16μl
  10. 1mol/L Tris-HCl, pH7.5・・・・160μl
  11. Ethachinmate・・・・・・・・・ 24μl
  12. 10mol/L Ammonium Acetate・・・960μl
  13. 3'Adaptor(50pmol/μ L)・・・・・ 8μl
  14. 5'Adaptor(50pmol/μ L)・・・・・ 8μl
  15. RT Primer(50pmol/μ L)・・・・・ 8μl
  16. 5'PCR Primer(50pmol/μ L)・・・ 16μl
  17. 3'PCR Primer(50pmol/μ L)・・・ 16μl
  18. Control RNA(30ng/μ L)・・・・・ 8μl


Please use Single Strand DNA Ligase, Thermostable Recombinant Solution (sold separately)from EPICENTRE with microRNA Cloning Kit Wako . Buffer components and reaction conditions are all optimized for this enzyme, which can be used in ligation reactions for both DNA and RNA (ATP-dependent). Optimal reaction temperature is 55- 65℃, enabling significantly more efficient adaptor ligation of microRNA than with T4 RNA ligase.

Outline

image


Code No. Description Package size
290-66501 freezemicroRNA Cloning Kit Wako 8 reactions


Target mRNA Cloning Kit Wako

Target mRNA Cloning Kit Wako is used for amplification of cDNA from target mRNA.
This kit is used for generating cDNA from mRNA interacting with microRNA in RNA fractions in immunoprecipitated Ago protein. DNA sequences obtained from cDNA synthesized from this mRNA and then cloned can be used for screening target mRNA candidates of microRNA. Using this kit in combination with Ago immunoprecipitation enables screening of target mRNA of microRNA not with previous database-based prediction, but with molecular biology-based methods.



Principles

image

Kit contents (10 reactions)

  1. dT(20)-RT Primer(20pmol/μL)・・・・・・・・・・10μL×1
  2. 10×RT Buffer・・・・・・・・・・・・・・・・・・20μL×1
  3. dNTP Mixture Solution (2.5mmol/l each)・・・・・20μL×1
  4. RNase Inhibitor(20U/μL)・・・・・・・・・・・・・10μL×1
  5. Reverse Transcriptase(200U/μL)・・・・・・・・・10μL×1
  6. 0.5 mol/L EDTA Solution・・・・・・・・・・・・・20μL×1
  7. 1mol/L Tris-HCl Solution・・・・・・・・・・・・・200μL×1
  8. Ethachinmate・・・・・・・・・・・・・・・・・・30μL×1
  9. 10mol/L Ammonium Acetate Solution・・・・・・480μL×1
  10. 2nd Strand Synthesis Primer(20pmol/μL)・・・・10μL×1
  11. 5' PCR Primer(20pmol/μL)・・・・・・・・・・・10μL×1
  12. 3' PCR Primer(20pmol/μL)・・・・・・・・・・・10μL×1
  13. 5' Colony PCR Primer(5pmol/μL)・・・・・・・・960μL×1
  14. 3' Colony PCR Primer(5pmol/μL)・・・・・・・・960μL×1


Code No. Description Package size
298-68001 freezeTarget mRNA Cloning Kit Wako 10 reactions
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Microarray Analysis

Toray 3D-Gene®

Please send us an RNA sample, and we will perform mRNA or microRNA gene expression analysis using they 3D-Gene®chip. We also offer consultations on statistical or biological analysis services by using analyzed data.

  • High sensitivity, reproducibility, and accuracy compared to traditional methods
  • Low noise through black resin and 3D pillar probe structure
  • Surface covered on nano-level with dense layer of oligo DNA probes
  • Promotes hybridization reaction with microbead agitation
image
image 3D-Gene®characteristics make it perfect for analysis of low-expression genes and microRNA, as well as analysis of RNA samples from small specimens, blood, or FFPE (formalinfixed paraffin embedded).
*Our services begin at RNA extraction step for blood and FFPE samples.
Method All human genes All mouse genes All rat genes miRNA chip Custom chip
DNA chip DNA chip DNA chip Human, mouse, rat (Creation and analysis services)
2-color -
1-color

*The microRNA chip is available only in 1-color.
* Please inquire about samples FFPE (formalin fixed paraffin embedded) samples or blood samples



■ Sample

Sample Total RNA Tissue/cell
Sample quantity 2 μg or more Please inquire.
Sample concentration purity 0.5 μg/μL or more (recommended)
OD260/OD280=1.8~2.0

■ Turnaround
Turnaround is usually within 1 month, but may differ according to sample quality(In the case of Japan).
■ Price
Please inquire.
■ How to order
 Please inquire through the Wako website:
 http://www.wako-chem.co.jp →Reagents → Services → Microarray analysis → 3D-Gene®


■ Notes

  • Please use recommended kits for RNA extraction.
  • Recommended kits differ for gene expression analysis and microRNA analysis.
  • Please send RNA samples dissolved in frozen DNase/RNase-free water, and use a frozen shipping services. Inquire first to determine a delivery time to set. We cannot be responsible for shipping-related problems.
  • We may not be able to accept highly infectious samples.
  • Please obtain informed consent from subject for any human samples.
  • We can only accept P1 level samples as determined by the Ministry of Education, Culture, Sports, Science and Technology's Act on the Conservation and Sustainable Use of Biological Diversity through Regulations on the Use of Living Modified Organisms.
  • Samples fundamentally cannot be returned.
  • This analysis service is intended for research use only. Please do not use for other purposes (medical diagnosis or drugs, food manufacture, testing, etc.)
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Wako Pure Chemical Industries, Ltd. Publication list

hAgo2(4G8) MAb
Cell. 2014 Jan 16;156:146-157.
Inefficient SRP interaction with a nascent chain triggers a mRNA quality control pathway.
microRNA Isolation Kit human / mouse Ago1, and human Ago2
Nucleic Acids Research , 2014,1-13.
Novel functional small RNAs are selectively loaded onto mammalian Ago1.
microRNA Cloning Kit Wako, ssDNA Ligase, thermostable
Mol Cell. 2011 Nov 4;44(3):424-36.
MCPIP1 Ribonuclease Antagonizes Dicer and Terminates MicroRNA Biogenesis through Precursor
MicroRNA Degradation.
Suzuki HI, Arase M, Matsuyama H, Choi YL, Ueno T, Mano H, Sugimoto K, Miyazono K.
Ago1(2A7), hAgo2(4G8), hAgo3(1C12) MAb
RNA Biol. 2011 Jan 1;8(1).
Deep-sequencing of human Argonaute-associated small RNAs provides insight into miRNA sorting and
reveals
Argonaute association with RNA fragments of diverse origin.
Burroughs AM, Ando Y, Hoon ML, Tomaru Y, Suzuki H, Hayashizaki Y, Daub CO.,
hAgo2(4G8) MAb
PNAS 2011 Mar 29.
MicroRNA let-7 establishes expression of {beta}2-adrenergic receptors and dynamically down-regulates
agonist-promoted down-regulation.
Wang WC, Juan AH, Panebra A, Liggett SB.
mAgo2(2D4) MAb
EMBO J . 2011 March 2; 30(5): 823–834.
Small RNA-mediated regulation of iPS cell generation
Zhonghan Li,Chao-Shun Yang, Katsuhiko Nakashima, and Tariq M Rana
mAgo2(2D4) MAb
Molecular Pharmacology March 22, 2011
RISC bound siRNA is a determinant of RNAi mediated gene silencing in mice.
Wei J, Jones J, Kang J, Card A, Krimm M, Hancock P, Pei Y, Ason B, Payson E, Dubinina N, Cancilla M,
Stroh M, Burchard J, Sachs A, Hochman J, Flanagan WM, Kuklin N.
mAgo2(2D4) MAb
Nature structural & molecular biology 2011, 18, 2, Feb
Genome-wide identi­ cation of Ago2 binding sites from mouse embryonic stem cells with and without
mature microRNA.
Leung AK, Young AG, Bhutkar A, Zheng GX, Bosson AD, Nielsen CB, Sharp PA.
Ago1(2A7), hAgo2(4G8), hAgo3(1C12) MAb
microRNA Isolation Kit, Ago1, Human Ago2 and Human Ago3

Genome Res. August 18, 2010
A comprehensive survey of 39 animal miRNA modi­ cation events and a possible role for 39 adenylation
in modulating miRNA targeting effectiveness.
Burroughs AM, Ando Y, de Hoon MJ, Tomaru Y, Nishibu T, Ukekawa R, Funakoshi T, Kurokawa T,
Suzuki H, Hayashizaki Y, Daub CO.
hAgo2(4G8) MAb
Nucleic Acids Research , 2009, 1–13
Expanded RNA-binding activities of mammalian Argonaute 2.
Tan GS, Garchow BG, Liu X, Yeung J, Morris JP 4th, Cuellar TL, McManus MT, Kiriakidou M.

hAgo2(4G8) MAb
Nature structural & molecular biology 2009 Dec 16;12:1259
Distinct passenger strand and mRNA cleavage activities of human Argonaute proteins.Wang B, Li S, Qi
HH, Chowdhury D, Shi Y, Novina CD
microRNA Isolation Kit, Human Ago2
BIOLOGY OF REPRODUCTION 2009 81, 717–729
Human Villous Trophoblasts Express and Secrete Placenta-Speci­ c MicroRNAs into Maternal Circulation
via Exosomes.
Luo SS, Ishibashi O, Ishikawa G, Ishikawa T, Katayama A, Mishima T, Takizawa T, Shigihara T, Goto T,
Izumi A, Ohkuchi A, Matsubara S, Takeshita T, Takizawa T.
hAgo2(4G8) MAb
BMC Research Notes 2009, 2:169
A useful approach to total analysis of RISC-associated RNA.
Hayashida Y, Nishibu T, Inoue K, Kurokawa T.
hAgo2(4G8) MAb
RNA 2009, 15:1078–1089
Mammalian GW182 contains multiple Argonaute-binding sites and functions in microRNA-mediated
translational repression.
Takimoto K, Wakiyama M, Yokoyama S.
mAgo2(2D4) microRNA Isolation Kit, Human Ago2
Reproduction 2008 Dec;136(6):811-22.
MicroRNA (miRNA) cloning analysis reveals sex differences in miRNA expression pro­ les between adult
mouse testis and ovary.
Mishima T, Luo SS, Ishibashi O, Kawahigashi Y, Mizuguchi Y, Ishikawa T, Mori M, Kanda T, Goto T,
Takizawa T.
hAgo2(4G8) MAb
Methods Mol Biol. 2008;442:29-43.
In vitro RNA cleavage assay for Argonaute-family proteins.
Miyoshi K, Uejima H, Nagami-Okada T, Siomi H, Siomi MC.
hAgo2(4G8) MAb
Nature , 2008 Sep 18;455(7211):421-4.
Prolyl 4-hydroxylation regulates Argonaute 2 stability.
Qi HH, Ongusaha PP, Myllyharju J, Cheng D, Pakkanen O, Shi Y, Lee SW, Peng J, Shi Y.
hAgo2(4G8) MAb
PNAS 2008 June 10, vol. 105 no. 23 7964–7969
Characterization of endogenous human Argonautes and their miRNA partners in RNA silencing.
Azuma-Mukai A, Oguri H, Mituyama T, Qian ZR, Asai K, Siomi H, Siomi MC.
hAgo2(4G8) MAb
J Hepatol. 2009 Mar;50(3):453-60. Epub 2008 Jul 9.
Regulation of the hepatitis C virus genome replication by miR-199a.
Murakami Y, Aly HH, Tajima A, Inoue I, Shimotohno K.


and others...........

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Products List

Other Reagents for microRNA Research

Code No. Description Package size
317-07363 refrigerator ISOGEN ® II 10ml
311-07361 100ml
315-02504 refrigeratorISOGEN ® 10ml
317-02503 50ml
311-02504 100ml
317-02623 refrigerator ISOGEN ® -LS 10ml
311-02621 100ml
314-07513 refrigerator ISOGEN ® with Spin Column 10 extractions
318-07511 60 extractions
194-15881 refrigeratorSuperSep™ RNA, 15%, 17well 5 gels
182-02711 freeze R-Mark™ small RNA Marker 50μl
311-03961 CLEAR STAIN Ag 20 stains
318-90041 5×TBE 1,000ml


Monoclonal Antibodies for microRNA Research

Code No. Description Package size
018-22401 refrigerator Anti Ago1, Monoclonal Antibody(1F2) 50μl
015-22411 refrigerator Anti Ago1, Monoclonal Antibody(2A7) 50μl
011-22033 refrigerator Anti Human Ago2, Monoclonal Antibody(4G8) 50μl
015-22031 100μl
014-22023 refrigerator Anti Mouse Ago2, Monoclonal Antibody(2D4) 50μl
018-22021 100μl
018-23241 refrigerator Anti Human Ago3, Monoclonal Antibody(1C12) 50μl
010-23821 refrigerator Anti Human Ago3, Monoclonal Antibody(6-107) 50μl
013-25491 -80 degree C Anti Mouse Ago3, Monoclonal Antibody(mA3-S11) 50μl
016-25501 -80 degree C Anti Mouse Ago3, Monoclonal Antibody(mA3-S9) 50μl
012-24741 -80 degree C Anti Ago4, Monoclonal Antibody(2B2) 50μl
019-24751 -80 degree C Anti Ago4, Monoclonal Antibody(2G7) 50μl
012-25581 -80 degree C Anti Ago1-4, Monoclonal Antibody(1G3) 200μl
018-25583 1ml
016-25584 5ml
017-23451 freeze Anti PIWIL1, Monoclonal Antibody(2C12) 100μl
018-23981 freeze Anti Human PIWIL2, Monoclonal Antibody(1A12) 50μl
015-23991 freeze Anti Human PIWIL2, Monoclonal Antibody(3C4) 50μl


Reagent Kits for microRNA Research

Code No. Description Package size
295-71701 refrigerator microRNA Extractor ® SP Kit 50 extractions
290-66501 freeze microRNA Cloning Kit Wako 8 extractions
298-68001 freeze Target mRNA Cloning Kit Wako 10 extractions
291-70201 refrigerator microRNA Isolation Kit, Human/Mouse Ago1 10 extractions
292-66701 refrigerator microRNA Isolation Kit, Human Ago2 10 extractions
292-67301 refrigerator microRNA Isolation Kit, Mouse Ago2 10 extractions
297-70301 refrigerator microRNA Isolation Kit, Human Ago3 10 extractions
295-74001 refrigerator MagCapture™ microRNA Isolation Kit, Human Ago2 10 extractions
297-74201 refrigerator MagCapture™ microRNA Isolation Kit, Mouse Ago2 10 extractions
293-74801 refrigerator MagCapture™ microRNA Isolation Kit, Ago1-4 10 extractions
299-74401 freeze MagCapture™ microRNA Isolation Kit, Protein G 20 extractions



・Listed products are intended for laboratory research use only, and not to be used for drug, food or human use.
・Please visit our online catalog to search for other products from Wako; http://www.e-reagent.com/
・This brochure may contain products that cannot be exported to your country due to regulations.
・Bulk quote requests for some products are welcomed. Please contact us.

QUOTATION REQUEST

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Laboratory Chemicals > microRNA Catalog

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