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RIP-Assay Kit for microRNA

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  1. What is the difference between RIP-Assay Kit for microRNA and RIP-Assay Kit?
    For the recovery of small RNAs, RIP-Assay Kit for microRNA should be used.
    RIP-Assay Kit is designed to recover large RNAs but not small RNAs.
    RIP-Assay Kit for microRNA is designed to recover both small RNAs and large RNAs. Moreover, by following different procedures, small RNAs and large RNAs can be isolated separately, or only small RNAs can be recovered. By recovering small RNAs which bind to RBPs/large RNAs, RIP-Assay Kit for microRNA enables you to study how the intracellular event of your interest is regulated by not only RBPs but also small RNAs. The kit is also useful for the identification of target RNAs that specifically bind to miRNAs of your interest.
  2. How many cells are needed to obtain small RNAs contained in RNP complexes?
    We recommend preparing 4×106 - 2×107 cells. However, appropriate cell number is different depending on the cell lines.
    For the first time users, it is recommended to use 1×107 cells/sample and optimize the cell number depending on the experimental conditions.
  3. What is the suitable RNA isolation method to obtain miRNAs most efficiently?
    If the contamination of large RNAs does not affect the following experiments, 2-step method is appropriate, since the recovery rates are high for both small RNAs and large RNAs. If the contamination of large RNAs should be avoided, isolate small RNAs by Separation method or 1-step method.
  4. How can miRNAs obtained with RIP-Assay Kit for microRNA be evaluated?
    To evaluate quality and quantity of obtained miRNAs, we recommend visualization of small RNAs using GelStar (Takara Bio, Inc.; code. F0535) or silver staining following RNA electrophoresis.
    After visualization, you can check the size, quantity and quality of obtained miRNAs compared to RNA ladder and control miRNAs. RNA quantitation reagents are also available if the obtained miRNAs are in the measurement range of the reagents.
    For downstream analysis of the obtained RNAs, we recommend the followings.
    ・Northern blot or RT-PCR for specific miRNAs
    ・RT-PCR, microarray or sequencing to identify miRNAs
  5. Besides gel extraction, what method can be used to isolate miRNAs?
    Depending on the following analysis, suitable isolation methods are different.
    For microarray analysis, the eluate can be used without any purification.
    For cloning or sequencing, miRNAs are recovered after the adapter ligation to miRNAs on beads.
    Kits such as small RNA Cloning Kit (Takara Bio, Inc.; code. RR065) are also available.
  6. Why do you perform both analyses with Bioanalyzer and silver staining to check the quality of recovered RNAs?
    With RIP-Assay Kit for microRNA, both large RNAs and small RNAs can be recovered. With Bioanalyzer, we examine whether the large RNAs obtained by RIP are degraded or intact. By checking the wave patterns of each RBP, we also analyze whether the obtained RNAs are specific to each RBP.
    Recovery of small RNAs is examined by silver staining, since small RNAs are undetectable by NanoDrop or Bioanalyzer.
  7. Are RNAs stabilized sufficiently by adding protease inhibitors without cross-linking?
    Protease inhibitors are added for the purpose of avoiding degradation of RBPs and antibodies, but not for the stabilization of RNA-RBP interactions. When you are planning to recover RNAs having only weak interaction with RBPs, cross-linking is necessary.
  8. Does formaldehyde (formalin) cross-linking affect the RNA isolation with RIP-Certified antibodies?
    We do not examine the influence of formaldehyde cross-linking. By using formaldehyde, protein-protein interactions as well as nucleic acid-protein interactions are cross-linked. Therefore, we think formalin is not very suitable for RIP-Assay after cross-linking.
  9. I would like to use magnetic beads. Is it possible?
    Dynabeads® (Invitrogen) can be used for Protein A and Protein G. You can also use Protein G-Magnetic Beads (MBL; code. MJS002V2).