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miRNA detection kit

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Product Description

  • Description
  • Experimental
  • Usage
  • Application
  • Ordering
The Hairpin-itTM miRNAs qPCR Quantitation Kit is a sensitive and specific method using real-time PCR for the detection and quantification of miRNAs (miRNA) from total RNA samples. MiRNAs are small, single-stranded, ~19–23 nt RNA molecules encoded in the genomes of plants, animals, and viruses. Mature miRNAs enter the RNA-induced silencing complex (RISC) and guide the RISC to induce translational repression or endonucleolytic cleavage of specific target mRNAs. Unlike commonly used methods for detection of miRNAs, the Hairpin-itTM miRNAs qPCR Quantitation Kit is more rapid and sensitive. 


This Kit contains miRNA specific RT and PCR primer set, with a Molecular Beacon probe included. The stem-loop like miRNAs RT primer and the miRNAs specific Beacon probe ensure the RT and PCR reaction would not be interfered by the miRNAs precursors. The reaction template could be total RNA or cell lysates.


To normalize for RNA content among different experimental samples, the U6 snRNA qPCR Normalization Kit (Cat # QPM-050) and 5S rRNA qPCR Normalization Kit (Cat # QPM-060) are available separately.
 
Hairpin-itTM miRNAs qPCR Quantitation Kit Reagents box includes the following items. Store the components at –20℃. Reagents must be stored in the dark.


Reagent
Amount
Storage
 
100rxns
200rxns
500 rxns
 
Real-time PCR Master Mix(5×)*
1.0 ml
2×1.0 ml
5×1.0 ml
4℃
miRNA specific primer Set(10µM)
100μl
200μl
1.0 ml
-20℃
miRNA RT primer(10µM)
15μl
50μl
100μl
-20℃
Taq DNA polymerase (5U/µl)
50μl
100μl
200μl
-20℃
Synthetic miRNA Standard
1nmol
 
 
-20℃
1X RNA Dilution buffer
2×1.0 ml
4×1.0 ml
10×1.0 ml
4℃
*Include dNTP Mixture,Mg2, Molecular Beacon probe.

 
Accessory Products

Some of the reagents supplied in the Hairpin-itTM miRNAs qPCR Quantitation Kit as well as other products suitable for use with the kit are available separately from Genepharma. Ordering information is provided below.

Item
Amount
Catalog no.
U6 snRNA qPCR Normalization Kit
20 rxns
50 rxns
QPM-090
QPM-091
5S rRNA qPCR Normalization Kit
25μl×50 rxns
25μl×100 rxns
QPM-080
QPM-081
miRNAs qPCR Quantitation Core Kit
100 rxns
200 rxns
500 rxns
QPM-030
QPM-031
QPM-032
Custom miRNAs Quantitation Kit
40µl×50 rxns
40µl×100 rxns
40µl×200 rxns
QPM-040
QPM-041
QPM-042


       

 

Hairpin-itTM miRNAs qPCR Quantitation Kit User Manual

 

Hairpin-itTMmiRNAs qPCR Quantitation Kit Reagents box includes the following items. Store the components at –20. Reagents must be stored in the dark.

                                                                 

 

Accessory Products


Some of the reagents supplied in the Hairpin-it
TM miRNAs qPCR Quantitation Kit as well as other products suitable for use with the kit are available separately from Genepharma. Ordering information is provided below. For more information, refer to our Web site (www.genepharma.com).


                                                               

 
 
Performing miRNAs RT Reaction
 
 
Introduction

The   Hairpin-itTM    miRNAs   real-time   Quantitation   assay   of miRNAs includes two steps, stem–loop RT reaction and real-time PCR detection. Stem–loop RT primers bind to at the 3’ end of miRNA molecules and are reverse transcribed with reverse transcriptase. Then, the  RT  product  is  quantified  using  real-time  PCR  that  includes miRNA-specific forward primer, reverse primer and a dye-labeled Molecular Beacon probes.
To exactly quantifying the copy number of miRNA in a certain RNA sample, a standard curve should be established.
To profiling miRNA’s expression relative to a said house-keeping gene, U6 snRNA or 5S rRNA qPCR Normalization Kit should be needed, see page 1 “Accessory products ”. For more detailed information    about      these       two  kits, please      log   on                    http:// www.genepharma.com.

 

 
 
Handling the synthetic miRNA
standard

The synthetic miRNA standard is supplied as 1μmol dry powder Oligo  stock.  Following  the  guideline  below  when  handling  the synthetic miRNA standard stock.Dilution: Centrifuge the tube at 10000rpm/min for 1 minute.Carefully open the tube, add 1ml RNA dilution buffer(supplied by the kit) to dilute the stock to 1nM and get another DEPC treated,steriled tubes to dilute the 1nM stock to
0.1nM for working concentration.Then prepare some other DEPC treated, steriled 1.5ml tubes to dilute the 1nM miRNA to serial orders (5~7 orders) to make the standard curve required for experiment.
Storage: It is important to store the 1nM miRNA standard solution and the 0.1nM work concentration solution at –20℃. And it is preferred to store the solution at –70℃ for longer time.RNase-free conditions: Take precautions to ensure that the stock solution does not become contaminated with RNase.
a. Use RNase-free steriled pipette tips and supplies for all manipulations.
b. Wear gloves when handling reagents and solutions.


 

Handling the miRNA RT primer

 

The miRNA RT primer is supplied as 10μM and please dillute it to 1μM working concentration according to the amount that you need in an experiment.Then store the 1μM miRNA RT primer solution at
–20℃. For a standard miRNA RT reaction, the final concerntration of RT primer is 50 nM.
 
 
Preparing The RT Reaction Mix

The template for the Hairpin-itTM  miRNAs real-time Quantitation assay can be total RNA, cell lysate or purified miRNA. The RNA template’s input range can vary from 0.2 ng to 200 ng or more as far as the requirement of the experiment. Other than the stability at 42℃, there is no special requirement of Reverse Transcriptase for the RT reaction. We recommended a standard 20μl
RT reaction size.More information can rely on the user manual of your own
RT reagents. The following table provides RT reaction Mix volumes for a standard 20μl reaction size.


1See the Important note on handling the RT primer.
2Add 1 pg to 1μg total RNA or mRNA to each reaction.
3Mix the RT reaction reagents(except RTase) and Mix by flipping the reagent tubes and pipette Mix for several times before RT reaction.Remember not to vortex.

Performing miRNAs RT Reaction

Standard RT Reaction Program
30 min at 16_℃,30 min at 42_℃,10 min at 85_℃
Keep all components, reaction mixes and samples on ice. After assembly, transfer the reaction mixes to a thermal cycler preheated to the cDNA synthesis temperature and begin RT reaction.Please take out the RT product quickly and put them on ice as soon as possible.The following procedures must be carried out at this low temperature all the time.Always keep in mind that do not take cDNA away from ice.
 
Handling the miRNA RT product

For the Hairpin-itTM  miRNAs real-time Quantitation assay, dilute the RT product for a proper ratio first (generally 3~4 times).Then mix it and pipette 2μl(20μl size) or 4μl(40μl size) miRNA RT reaction product as the template for real-time PCR step subsequently. Store the surplus miRNA RT reaction product at –20℃.
 
Preparing Real-Time PCR Reaction Mix

The following table provides Real-time PCR reaction Mix volumes for a 20μl and 40μl reaction size. Note that preparation of a master mix is crucial  in  quantitative  applications  to  reduce  pipetting  errors  in  40μl reaction size.
 
1The 1×Buffer contains 3mM Mg2+, 0.2mM dNTP,SYBR Green dye.
2The miRNA specific Primer set includes PCR forward and reverse primers.
 
 
For multiple reactions, prepare a master mix of common components, add the proper volume to each tube or plate well, and then add the unique action components.
Program the real-time PCR instrument to perform PCR amplification as shown below.
95℃ for 3 minutes’ hold, 40 cycles of:
95℃, 12 seconds
62, 40~60 seconds
You should select FAM or SYBR for detection and ROX for reference dye and detection step is at 62.

The synthetic miRNAs standard provided by this kit can work as a miRNA control when spiked into the total RNA extracted from tissue or cells. If the exact copy number of the miRNA in a certain amount RNA sample was wanted, you can
construct a standard curve follow the recommendation step below:
 

Constructiona standard curve

1. Add 1ml RNA dilution buffer to dilute the synthetic miRNAs standard to 1nM
stock. Prepare another 1.5ml tube to dilute the 1nM miRNA standard solution to
0.1nM as work concentration to a probably volume.and stored 1nM stock solution separated from primers and Master Mix at -70℃.
2. Dilute the 0.1nM synthetic miRNAs to 5~7 order of magnitude with RNase free
H2O.
3. Add 2μl RNA sample dilution to 20μl RT reaction system. Note that each order should be prepared for 2 or 3 repeats in RT reaction.
4. Add 4μl RT reaction product to 40μl real-time PCR system. Note that each order should also be prepared for 2 or 3 repeats in PCR reaction.
5. For the standard curve, the 2μl 0. 1nM synthetic miRNAs sample added into 20μl
RT reaction system represents 6 ×107 copies per reaction.

 
1. For multiple reactions, prepare a master mix of common components, add the appropriate volume to each tube or plate well on ice, and then add the unique reaction components (e.g., template). Preparation of a master mix is crucial in qRT-PCR to reduce pipetting errors.

2. Make sure that all components are at the bottom of the tube/plate; centrifuge briefly if needed.


 
  1. Cui, W., Q. Li, et al. "MiR-126-3p regulates progesterone receptors and involves development and lactation of mouse mammary gland." Molecular and Cellular Biochemistry 355(1): 17-25.

  2. Zhang, C., J. Zhang, et al. "High level of miR-221/222 confers increased cell invasion and poor prognosis in glioma." Journal of Translational Medicine 10(1): 119.

  3. Li, H. M., C. M. Wang, et al. "MiR-15a Decreases Bovine Mammary Epithelial Cell Viability and Lactation and Regulates Growth Hormone Receptor Expression." Molecules 17(10): 12037-12048.

  4. Chen, L., L. Han, et al. "VHL regulates the effects of miR-23b on glioma survival and invasion via suppression of HIF-1/VEGF and -catenin/Tcf-4 signaling." Neuro-Oncology 14(8): 1026-1036.

Ordering
The first problem we meet for the application of siRNA is to design the sequences. Although there are quite a lot principles for the designing of published sequences and quite a few companies supply on-line design service, up to now, no software can guarantee the efficiency of the oligos designed.
Shanghai GenePharma Co, Ltd. and our collaboration laboratories have been involved in RNAi research for some time. For each specific gene target, if 4 pairs of oligos were designed and synthesized, normally you will have at least one pair showing 70% down regulation.
Our price: 2OD, PAGE purified, $89/pair
*If one single order is less than 5 pairs extra shipping fee of $40 will be charged. Payment terms are 30 days. We accept cheque or wire transfer into our USD account.

 Specifications of siRNA Oligos:
Quality Control:
All our siRNA oligos undergo vigorous process monitoring and strict quality control. Length and labeling are systematically controlled by PAGE or mass spectrometry analysis. Quantity is systematically validated by UV absorbance at 260 nm.
Purification: Fully deprotected and desalted.
Purified by PAGE or RP-HPLC upon request
Length: 19 to 23 mers
Bases: RNA (A, C, G or U)
DNA (A, C, G or T)
Backbone: Phosphodiester bond
Labels and modifications: Fluorescein, biotin and phosphate: 3' or 5'end
Other labels available upon request
Format: Single-strand RNA oligos is delivered in dry form
Storage and stability
Although oligonucleotides are stable in solution at 4°C for up to 2 weeks, we recommend storage should be at -20°C. Repetitive freeze-thaw cycles should be avoided by storing as aliquots. For long-term storage, siRNA oligos should be dried.
Oligonucleotides with fluorescent labels should be protected from light. We guarantee our oligonucleotides for six months, when stored under the above conditions.
Shipment: Shipped by express delivery, dry in individual, transparent tubes.
Oligonucleotide Technical Data Sheet

Oligonucleotides are delivered with an Oligonucleotide Technical Data Sheet, which includes oligonucleotide name, sequence, concentration, size,  purification method.

Services available upon request

1.     Aliquoting

2.     Free design support

3.     Additional services may increase turn-around time

Ordering:Please fill in the following order form and send it to: order@genepharma.com or by fax to:00862151320295 our staff will confirm with you asap.