The Mouse Endoribonuclease Dicer (DICER1) ELISA Kit is a powerful tool designed for the accurate measurement of DICER1 levels in mouse serum, plasma, and cell culture supernatants. This kit offers exceptional sensitivity and specificity, ensuring precise and consistent results, making it ideal for various research applications.DICER1 is a key enzyme involved in the processing of microRNA molecules, playing a critical role in gene regulation and RNA interference. Dysregulation of DICER1 has been implicated in various diseases, including cancer, autoimmune disorders, and neurological conditions, highlighting its importance as a biomarker for disease research and therapeutic development.
With its reliable performance and versatility, the Mouse Endoribonuclease Dicer (DICER1) ELISA Kit is a valuable tool for scientists studying molecular mechanisms, disease pathways, and potential therapeutic targets in mouse models. Trust in this kit to deliver accurate and consistent results for your research needs.
Isoform 2: More active than isoform 1 to process long double-stranded RNA into siRNAs. Responsible for the accumulation of endogenous siRNAs observed in mouse oocytes compared to somatic cells and it regulates meiotic spindle organization in female germline.
Uniprot:
Q8R418
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant mouse Endoribonuclease Dicer
Sub Unit:
Component of the RISC loading complex (RLC), or micro-RNA (miRNA) loading complex (miRLC), which is composed of DICER1, AGO2 and TARBP2; DICER1 and TARBP2 are required to process precursor miRNAs (pre-miRNAs) to mature miRNAs and then load them onto AGO2. Note that the trimeric RLC/miRLC is also referred to as RISC. Interacts with DHX9, AGO1, PIWIL1 and PRKRA. Interacts with AGO2, TARBP2, EIF6, MOV10 and RPL7A (60S ribosome subunit); they form a large RNA-induced silencing complex (RISC). Interacts with BCDIN3D.
Subcellular Location:
Cytoplasm
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
DICER1: a cytoplasmic type-III RNase that plays a central role in RNA interference (RNAi) pathways to produce the active small RNAs that represses gene expression. Possesses a DEAD box RNA helicase motif in its amino terminus and a dsRNA-binding motif in the carboxy terminus. Dicer, along with Ago2 andTRBP, are the main components of the RNA-induced silencing complex (RISC). Fragile X syndrome repeats form RNA hairpins that are cut by Dicer but do not activate the interferon-inducible protein kinase, PKR. Four alternatively spliced isoforms have been described.Protein type: Helicase; Cell development/differentiation; Ribonuclease; RNA-binding; EC 3.1.26.3; RNA processingCellular Component: axon; cytoplasm; dendrite; ER-Golgi intermediate compartment; growth cone; nucleusMolecular Function: ATP binding; deoxyribonuclease I activity; DNA binding; double-stranded RNA binding; endonuclease activity; endoribonuclease activity; endoribonuclease activity, producing 5'-phosphomonoesters; helicase activity; hydrolase activity; metal ion binding; miRNA binding; nuclease activity; nucleotide binding; protein binding; protein domain specific binding; ribonuclease III activity; RNA binding; siRNA bindingBiological Process: anatomical structure development; angiogenesis; branching morphogenesis of a tube; cardiac muscle cell development; cartilage development; cell proliferation; cerebral cortex development; defense response to virus; DNA fragmentation during apoptosis; embryonic hindlimb morphogenesis; embryonic limb morphogenesis; epidermis morphogenesis; gut development; hair follicle development; hair follicle morphogenesis; inner ear receptor cell development; lung development; meiotic spindle organization and biogenesis; miRNA-mediated gene silencing, miRNA loading onto RISC; miRNA-mediated gene silencing, production of miRNAs; mRNA stabilization; multicellular organism growth; myelin formation in the peripheral nervous system; myoblast cell differentiation involved in skeletal muscle regeneration; negative regulation of transcription from RNA polymerase II promoter; nerve development; neurite morphogenesis; olfactory bulb interneuron differentiation; positive regulation of myelination; positive regulation of Schwann cell differentiation; positive regulation of transcription from RNA polymerase II promoter; post-embryonic development; pre-microRNA processing; regulation of cell cycle; regulation of cell differentiation; regulation of gene expression; regulation of myelination; regulation of neurogenesis; regulation of neuron differentiation; regulation of Notch signaling pathway; regulation of odontogenesis of dentine-containing teeth; regulation of oligodendrocyte differentiation; regulation of protein amino acid phosphorylation; regulation of RNA metabolic process; regulation of viral genome replication; reproductive structure development; RNA interference, conversion of ds siRNA to ss siRNA; RNA interference, production of siRNA; RNA interference, siRNA loading onto RISC; RNA interference, targeting of mRNA for destruction; RNA processing; RNA-mediated gene silencing; rRNA catabolic process; spinal cord motor neuron differentiation; spindle assembly; spleen development; stem cell maintenance; zygote asymmetric cell division
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
Step
1.
Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.
Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.
Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.
Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.
Repeat the wash process for five times as conducted in step 3.
6.
Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.
Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.
Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.
After experiment, store all reagents according to the specified storage temperature respectively until their expiry.
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
Sample Type
Protocol
Serum
If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma
Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid
Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant
Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates
Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates
The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates
Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk
Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.
