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RIP Sequencing Q&A

  • General Questions

  • What information should I know before I do the RIP-Seq?
    • Before doing a RIP-SEQ you generally need to specify
      • The type of sample to be done
      • What RBPs are of interest
      • Whether IP-level antibodies are available for these RBPs
      • The type of RNA sequences bound by the RBPs of interest (e.g. mRNA, miRNA, lncRNA, circRNA, etc.)
  • What are the requirements for selecting endogenous IP for RIP-seq?
    • Usually for endogenous IP, there are some necessary conditions to be met - (i) there is an IP-grade antibody; (ii) the protein starting amount should be large enough.
  • How to choose the antibody for RIP?
    • For the low binding abundance of protein and RNA, usually the IP efficiency is not very high, the amount of RNA is small, and there is a greater risk of library building, so you can choose multiple IPs to increase the amount of product.
  • Why do WB pre-experiment evaluation before RIP experiment?
    • The main purposes of Western Blot are (i) to detect the specificity of the antibody and whether it can bind the target protein; (ii) to identify the target protein bands by mass spectrometry; and (iii) to detect whether the target protein is expressed in the sample and its abundance level.
  • My antibody has a target protein band for WB, but why no band for RIP?
    • There are many immunoassays with different purposes, such as WB, ELISA, IP, IF, ChIP, IHC(P), FACS, among which only WB is an experiment that requires denaturation of proteins. In this case, any antibody to this protein can recognize the antigen that becomes linear in the WB; however, antibodies that can do WB may not be used for non-denaturing tests of IP, IF, and IHC, because the three-dimensional structure of natural proteins hides many antibody binding sites, and WB-level antibodies cannot bind to them.
      To do RIP experiments, you need to buy IP-level antibodies.
  • Are there any quality control experiments that need to be done after immunoprecipitation in RIP experiments?
    • After immunoprecipitation, on the one hand, the enriched proteins are subjected to WB quality control to test the specificity of the IP and the enrichment effect of the target protein. On the other hand, RNA should be extracted for quantitative analysis and the sequencing library constructed should be quality checked.
  • What do I need to pay attention to in RIP experiments?
    • 1. Preventing non-specific binding of RNA to proteins
      2. Avoid disruption of RNA-protein binding
      3. Avoiding contamination by exogenous RNase
      4. Inhibit the activity of endogenous RNase
      5. Select antibodies suitable for RIP
      6. Avoid degradation of RNA-binding proteins.
  • How to evaluate the WB QC results after RIP experiments?
    • 1. If the target protein is expressed in the sample and the antibody is effective, the target protein can be detected in the sample and Input produces a band at the corresponding position.
      2. If the antibody used can effectively enrich the target protein, then IP will also produce a band at the corresponding position; in addition, there will also be antibody bands, which are due to the fact that the antibody is tightly bound to the protein and cannot be separated before the sample is loaded, and the heavy chain (or light chain) of the enzyme-labeled secondary antibody-conjugated antibody added during WB QC will produce a band at 55kD (or 25kD).
      3. IgG can help us to determine whether the immunoprecipitation system has non-specific binding to the protein or not, and the RIP result with high specificity should be no destination band and only heavy (or light) chain band of IgG.
  • How to discover miRNA by RIP-seq and study to predict its function?
    • miRNAs can regulate cellular processes related to neuronal function, including the formation of neurosynapses. We take the example of studying miRNAs in neurons. By constructing a GFP-tagged vector and injecting it into mouse brains, we obtained miRNA and target mRNA using RIP; and verified the RIP efficiency by qRT-PCR. Then the RNA fragments obtained by RIP were sequenced in high throughput and compared with the reference genome of mice and miRbase database respectively for annotation and GO function enrichment analysis, etc. to discover the functions of miRNAs.
  • Is the IP-enriched RNA the target protein-binding RNA?
    • Not exactly, RIP does enrich the target protein-binding RNA, so the coverage of sequencing reads is higher in the protein-binding regions of the reference genome, forming obvious peaks compared to other regions. However, due to the differences in RNA expression and the preferences introduced by the amplification and sequencing processes, the coverage of reads in non-enriched regions may also have high and low peaks, which undoubtedly interferes with the screening of binding peaks (Peak calling).

  • Sample Preparation

  • How to store the samples for RIP-seq?
    • After the samples are isolated and quickly cleaned and labeled, they should be immediately snap-frozen in liquid nitrogen for at least 30 minutes and then stored in a -80°C refrigerator or dry ice to ensure that the samples are always at -80°C before the experimental operation to avoid protein degradation.
  • Can the extracted RNA be used for RIP experiments?
    • Of course not, because RIP-Seq is performed by pulling down the RNA binding protein (RBP) with a specific antibody, which binds the RNA sequence, and then the bound RNA sequence is measured. There is no longer RBP in the extracted total RNA.
  • What is the amount of animal tissue sampled for RIP-seq? And how to sample?
    • Animal tissues
      1. Remove fresh tissue, which should exclude tissue types not required for the study, such as connective and adipose tissue.
      2. Quickly rinse the tissue surface with pre-chilled 0.9% saline to rinse off any residual blood.
      3. If the tissue is large, try to cut the tissue into small pieces ≤ 0.5 cm in length, width, and height (i.e., the size of a soybean).
      4. Mix the processed tissue samples well and store in a 10 ml or larger volume screw cap lyophilization tube, accurately labeled.
      5. Rapidly place in liquid nitrogen for 30 minutes and then transfer to -80°C or liquid nitrogen for storage.
      6. Transport: Place lyophilized tubes in 50 ml centrifuge tubes or in well-sealed plastic bags and transport on dry ice.
      The sample size for animal tissues
      1. Individual smaller animals such as mice, chicks, zebrafish, etc. liver, heart, brain and other tissues: >1g.
      2. Individual larger animals such as pigs, cattle, sheep, etc. muscle, late embryo and other tissues: >2g adipose tissue >10g.
      3. Whole individuals such as insects: >3g.
  • If studying tumor samples, what are the sample requirements for RIP-Seq?
    • 1. For tumor tissues, tumor and normal tissues should be determined as accurately as possible, and if possible please judge the sampling site to be studied based on the results of the frozen section report, and tumor tissues should be excised from surrounding normal tissues (normal tissues should also be excised from surrounding tumor tissues).
      2. Immediately after isolation, divide the tissue, approximately 2-3 mm thick, and place the tissue in a 2.0 ml or larger volume screw cap lyophilization tube.
      3. Rapidly freeze in liquid nitrogen for 30 minutes, then transfer to -80°C or liquid nitrogen for storage.
      4. Transport: Place the lyophilized tubes in 50 ml centrifuge tubes or small plastic bags with good sealability and transport on dry ice.
      5. Tumor tissue: >1 g.
  • Can plant tissue be used for RIP-SEQ? How many plant samples do I need to provide?
    • IP enrichment yields are low, and library building may fail if the starting amount required is not reached. Especially for plant tissues, the amount of samples sent must be guaranteed because plant leaves, fruits, tubers or rhizomes contain high concentrations of polysaccharide polyphenolic substances, as well as other complex unknown components, which are difficult to extract.
      1. From the plant body, fresh tissues are taken. For material obtained in the field or in the field, the dust or soil on the surface of the material needs to be rinsed off and blotted dry after extraction.
      2. If the tissue is large, it should be cut into small pieces as much as possible and then placed in a 10 ml or larger volume lyophilization tube, or wrapped tightly in tin foil and placed in a self-sealing bag, accurately labeled.
      3. Rapidly place in liquid nitrogen for 30 minutes and then transfer to -80°C or liquid nitrogen for storage.
      4. Sample volume. Strain: >5g. Root system: >5g. Stems: >5g. Leaves: >5g. Fruit: >10g. Seeds: >5g. Inflorescence, stamens, pollen: >2g.
  • My sample is cell, what do I need to do before doing RIP-Seq delivery?
    • 1. Cells in wall culture should be processed into cell suspension and washed twice with PBS buffer (prepared without Rnase), centrifuged at 3,000 rpm/min, 4 °C for 5 min, supernatant poured out and accurately labeled.
      2. Rapidly freeze in liquid nitrogen for 30 min, then transfer to -80 °C or store in liquid nitrogen.
      3. Transport: place the lyophilized tubes in 50 ml centrifuge tubes or small plastic bags with good sealability and transport on dry ice.
      4. Sample size: 1 x 108.
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