Human Endoribonuclease Dicer (DICER1) ELISA Kit

Product Name:	Human Endoribonuclease Dicer (DICER1) ELISA Kit
SKU:	HUEB0439
Size:	96T
Target:	Human Endoribonuclease Dicer (DICER1)
Synonyms:	Helicase with RNase motif, Helicase MOI, DICER, HERNA, KIAA0928
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.312-20ng/mL
Sensitivity:	0.11ng/mL

Intra CV:

5.2%

Inter CV:

8.1%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	110-119%	109-119%	86-96%	94-104%
EDTA Plasma(N=5)	103-111%	92-101%	89-102%	104-113%
Heparin Plasma(N=5)	107-119%	101-113%	93-105%	107-116%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	97	91-103
Plasma	99	93-105

Function:

Double-stranded RNA (dsRNA) endoribonuclease playing a central role in short dsRNA-mediated post-transcriptional gene silencing. Cleaves naturally occurring long dsRNAs and short hairpin pre-microRNAs (miRNA) into fragments of twenty-one to twenty-three nucleotides with 3' overhang of two nucleotides, producing respectively short interfering RNAs (siRNA) and mature microRNAs. SiRNAs and miRNAs serve as guide to direct the RNA-induced silencing complex (RISC) to complementary RNAs to degrade them or prevent their translation. Gene silencing mediated by siRNAs, also called RNA interference, controls the elimination of transcripts from mobile and repetitive DNA elements of the genome but also the degradation of exogenous RNA of viral origin for instance. The miRNA pathway on the other side is a mean to specifically regulate the expression of target genes.

Uniprot:	Q9UPY3
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human 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. Associates with the 60S ribosome. Interacts with BCDIN3D. Interacts with AGO2, TARBP2, EIF6, MOV10 and RPL7A (60S ribosome subunit); they form a large RNA-induced silencing complex (RISC) (PubMed:17507929).
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.
UniProt Protein Details:	Protein type:Helicase; Cell development/differentiation; Ribonuclease; RNA-binding; EC 3.1.26.3; RNA processing Chromosomal Location of Human Ortholog: 14q32.13 Cellular Component: growth cone; axon; cytoplasm; dendrite; ER-Golgi intermediate compartment; nucleus; cytosol Molecular Function:endoribonuclease activity; protein binding; miRNA binding; ribonuclease III activity; metal ion binding; double-stranded RNA binding; deoxyribonuclease I activity; helicase activity; ATP binding Biological Process: zygote asymmetric cell division; regulation of neuron differentiation; regulation of cell cycle; regulation of oligodendrocyte differentiation; miRNA-mediated gene silencing, miRNA loading onto RISC; negative regulation of transcription from RNA polymerase II promoter; embryonic hindlimb morphogenesis; olfactory bulb interneuron differentiation; post-embryonic development; cardiac muscle cell development; pre-microRNA processing; angiogenesis; DNA fragmentation during apoptosis; defense response to virus; regulation of viral genome replication; spleen development; positive regulation of myelination; positive regulation of Schwann cell differentiation; hair follicle morphogenesis; inner ear receptor cell development; mRNA stabilization; RNA interference, targeting of mRNA for destruction; multicellular organism growth; stem cell maintenance; RNA interference, conversion of ds siRNA to ss siRNA; spindle assembly; spinal cord motor neuron differentiation; miRNA-mediated gene silencing, production of miRNAs; myelin formation in the peripheral nervous system; branching morphogenesis of a tube; cartilage development; gene expression; nerve development; cerebral cortex development; RNA interference, production of siRNA; reproductive structure development; neurite morphogenesis; lung development; myoblast cell differentiation involved in skeletal muscle regeneration Disease: Pleuropulmonary Blastoma; Goiter, Multinodular 1, With Or Without Sertoli-leydig Cell Tumors; Rhabdomyosarcoma, Embryonal, 2
NCBI Summary:	This gene encodes a protein possessing an RNA helicase motif containing a DEXH box in its amino terminus and an RNA motif in the carboxy terminus. The encoded protein functions as a ribonuclease and is required by the RNA interference and small temporal RNA (stRNA) pathways to produce the active small RNA component that represses gene expression. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Sep 2010]
UniProt Code:	Q9UPY3
NCBI GenInfo Identifier:	257051056
NCBI Gene ID:	23405
NCBI Accession:	Q9UPY3.3
UniProt Secondary Accession:	Q9UPY3,O95943, Q9UQ02, A7E2D3, B3KRG4, E0AD28,
UniProt Related Accession:	Q9UPY3
Molecular Weight:	1922
NCBI Full Name:	Endoribonuclease Dicer
NCBI Synonym Full Names:	dicer 1, ribonuclease type III
NCBI Official Symbol:	DICER1
NCBI Official Synonym Symbols:	DCR1; MNG1; Dicer; HERNA; RMSE2; Dicer1e; K12H4.8-LIKE
NCBI Protein Information:	endoribonuclease Dicer; helicase MOI; Dicer1, Dcr-1 homolog; helicase with RNAse motif; dicer 1, double-stranded RNA-specific endoribonuclease
UniProt Protein Name:	Endoribonuclease Dicer
UniProt Synonym Protein Names:	Helicase with RNase motif; Helicase MOI
Protein Family:	Endoribonuclease
UniProt Gene Name:	DICER1
UniProt Entry Name:	DICER_HUMAN

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
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.

ELISA

Human Dicer ELISA Kit