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RNAi Screening service

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Product Description

Using siRNA screening to select the genes which related cells life cycle has become a common tool for the research of gene chemistry and life science. The high throughput screening could analyze the functions of thousands genes, and promptly identify the relative genes,thus find new research targets

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siRNA/miRNA high throughput screen service
Using siRNA screening to select the genes which related cells life cycle has become a common tool for the research of gene chemistry and life science. The high throughput screening could analyze the functions of thousands genes, and promptly identify the relative genes,thus find new research targets.

The siRNA libraries available

1)full human genome siRNAlibrary (21585 genes)
2)full human miRNA library and inhibitor (983×2)
3)cell cycle siRNAlibrary (131 genes)
4) Deubiquitinating enzymes siRNA library (127 genes)
5) phosphorylase library (257 genes)
6) cytokine receptor siRNA library (319 genes)
7) G-protein-coupled receptor siRNA library (516 genes)
8) apoptosis siRNA library (558 genes)
9) protein kinase siRNA library (779 genes)
10)custome siRNA library (100-2000 genes)

 

service procedures

1.Raise the opinions of siRNA screen: clients first land our web to understand our service content, and fill in the screen application form, and send it back to us, the clients also need to provide the background information of your target cell pathways.
2.confirm the screen plan. GenePharma’s research staffs will establish the screen plan based on the clients information. Clients will sign the contract with GenePharma and pay some proportion of front money.
3.optimize the screen condition. We will optimize the screen condition based on the information that the clients provided( cell type, migration character,so on)
cell transfection efficiency optimize is the key factor to conduct a success siRNA screen。
3.screen modle quantitive evaluation and optimize

First of all, find out a strong positive control and a stable negative control. The values of positive control and negative control should be stable between plates and different experiments. So that the value of Z’ could be analyzed and optimized to judge whether those two controls is suitable enough to conduct following large scale screening
 





Second, conduct high-throughput screening with positive control and negative control on the same plate every time.
Third, to select the positives, we first analyze the distribution of values accord to normal distribution, then we normalize the values and find out those greatly deviates from the list.
We perform the primary screening by a pool of 3 siRNAs. For these positives, the validation will be done by testing the individual siRNA of each gene. It is better to verify the result with a second cell assay.
4.small scale trail screen, once confirmed the cell screen condition, we will use the 354 well plates or SAMcell chips to screen about 100 genes and testify whether the result is reliable. 
5、.large scale siRNA library screen: TO conduct massive siRNA/miRNA screen on the target genes.
6、apply the screen report; after the massive screen, we will statistic and analyze the data, provide report to the clients, and descide the next steps。
7、 for the positive results, we will contact with the clients, and testify the positive results by chery pick-up method.
The whole screen process last about 3-6 months, the time will descide the scale of the library and screen plan.

Screen Technique


1. The screening will be performed based on Multi-well plates (384 well or 96 well), or on self-assembled cell microarray (SAMcell) (patented), in which 100 siRNAs are microprinted on a single covership. (Zhang H. et al. Nature Communication 2011 v2:554; Rong Y. et al, Nature Cell Biology 2012 v14(9):924-34).
2. We developed a unique approach for manufacturing esiRNA (Huang, H et al. Lab Chip, 2011; Wang, Z. et al. PLoS ONE, 2012).

For more information please check the siRNA screen guid manuals.

Our success siRNA screen examples










EnSpire® Multimode Plate Readers (PerkinElmer).

Success examples

  1. We finished the screening of functional genes regulating cell migration. The library we screened includes miRNA mimics libraries (~900) (Zhang H. et al. Nature Communication 2011 v2:554.) and Human kinase siRNA library (~750) (under review).
  2. We finished the siRNA screening of functional genes regulating autophagosome formation. The library we screened includes custom-based siRNA library (~300) (Rong Y. et al, Nature Cell Biology 2012 v14(9):924-34). The screen using the whole human genome siRNA library (ABI, ~20,000) is undergoing.
  3. We finished the screening of functional genes regulating cell invasion by use of multi-well plates. The library we screened includes miRNA mimics libraries (~900) and human kinase siRNA library (~750).
  4. We finished the screening of functional genes regulating cell apoptosis. The library we screened includes miRNA mimics libraries (~900), Human kinase siRNA library (~750) and esiRNAs(~100) (Wang Z. et al. PLoS ONE, 2012)
  5. We accomplished the screening of functional genes regulating mitochondrial fission and fusion. The library we screened includes miRNA mimics libraries (~900) (paper in preparation).
  6. We are screening functional genes regulating NADH metabolism. The library includes Human kinase siRNA dibry (~750).
  7. We are screening functional genes capable of generating synthetic lethal with a chemical. The library includes Human kinase siRNA library (~750) and phosphatase siRNA library (~300).
  8. We are performing the screen in other cell assays, including insulin resistance, modulators in TGF-beta pathway, etc.

Do you offer siRNA/shRNA screening services (yes/no)?
Yes

Please describe your previous experience regarding screening of siRNA, shRNA and/or ORF libraries. Please include type, number, format, and size of past libraries screened etc...

  1. We finished the screening of functional genes regulating cell migration. The library we screened includes miRNA mimics libraries (~900) (Zhang H. et al. Nature Communication 2011 v2:554.) and Human kinase siRNA library (~750) (under review).
  2. We finished the siRNA screening of functional genes regulating autophagosome formation. The library we screened includes custom-based siRNA library (~300) (Rong Y. et al, Nature Cell Biology 2012 v14(9):924-34). The screen using the whole human genome siRNA library (ABI, ~20,000) is undergoing.
  3. We finished the screening of functional genes regulating cell invasion by use of multi-well plates. The library we screened includes miRNA mimics libraries (~900) and human kinase siRNA library (~750).
  4. We finished the screening of functional genes regulating cell apoptosis. The library we screened includes miRNA mimics libraries (~900), Human kinase siRNA library (~750) and esiRNAs(~100) (Wang Z. et al. PLoS ONE, 2012)
  5. We accomplished the screening of functional genes regulating mitochondrial fission and fusion. The library we screened includes miRNA mimics libraries (~900) (paper in preparation).
  6. We are screening functional genes regulating NADH metabolism. The library includes Human kinase siRNA library (~750).
  7. We are screening functional genes capable of generating synthetic lethal with a chemical. The library includes Human kinase siRNA library (~750) and phosphatase siRNA library (~300).
  8. We are performing the screen in other cell assays, including insulin resistance, modulators in TGF-beta pathway, etc.

If you provide screening, please highlight your key equipment and facility capabilities (BSL level). Please detail type of equipment, brand, model and quantity etc…Please also detail any methods used to select and validate positives.

Equipment and facility capabilities

  1. The Phoenix Liquid Handling System (Art Robinns Instruments): 96 or 384 channel positive displacement syringe head. Dispensing volumes down to 100 nL.
  2. The Agilent Bravo Automated Liquid Handling Platform (Agilent): 96 or 384 channel. Dispensing volumes down to 2 µL.
  3. MultidropCombi Reagent Dispenser (Thermo Scientific ): 96 or 384 well plate, dispensing volumes in the range of 0.5 to 2500 µL.
  4. ImageXpress Micro Widefield High Content Screening System (Molecular Devices): capture research-quality, widefield images of fixed- or live-cell assays.
  5. EnSpire® Multimode Plate Readers (PerkinElmer).
  6. All the molecular and cellular culture instruments
  7. 2,000 square feet clean room (Biosafety level 2), and 3,000 square feet Class 10,000 room

Screening Methods
1. The screening will be performed based on Multi-well plates (384 well or 96 well), or on self-assembled cell microarray (SAMcell) (patented), in which 100 siRNAs are microprinted on a single covership. (Zhang H. et al. Nature Communication 2011 v2:554; Rong Y. et al, Nature Cell Biology 2012 v14(9):924-34).
2. We developed a unique approach for manufacturing esiRNA (Huang, H et al. Lab Chip, 2011; Wang, Z. et al. PLoS ONE, 2012).

Methods used to select and validate positives
First of all, find out a strong positive control and a stable negative control. The values of positive control and negative control should be stable between plates and different experiments. So that the value of Z’ could be analyzed and optimized to judge whether those two controls is suitable enough to conduct following large scale screening
 
Second, conduct high-throughput screening with positive control and negative control on the same plate every time.
Third, to select the positives, we first analyze the distribution of values accord to normal distribution, then we normalize the values and find out those greatly deviates from the list.
We perform the primary screening by a pool of 3 siRNAs. For these positives, the validation will be done by testing the individual siRNA of each gene. It is better to verify the result with a second cell assay.

What is your current quarterly capacity (number of studies and samples)?
We have six full-time employees. We are able to perform 3 or 4 studies in parallel, totally 50 multi-well plates (96 or 384) every day. Thus, more than a dozen studies can be accomplished in the scale of 2,000 siRNAs in each quarter.

Please indicate any requirements or other upfront requirements for these services.
It would be better to know cell assay, cell types, transfection condition or signal types.

Please specify any unique capabilities or advantages your organization may possess in this area.

1. The patented self-assembled cell microarray (SAMcell) technology is suitable for almost all image-based screening with the following advantages.
A. Low cost: The cell number is small, so that the siRNA, assay reagent including transfection reagents and antibody is low.
B. Low background: al lcell islands grow on the same environment unlike multiwall plate.
C. Easily tracking cell behaviors (real time or fixed).
2. Skilled and experienced team with HTS technology services, including assay development, screening, data collection and analysis, as well as validation.
3. More than ten efficient and diverse consultants from Peking University, Tsinghua University, Harvard University, etc..

Please describe and provide a breakdown of any costs of associated with siRNA/shRNA library screening.

Item

Price

Notes

Cell assay development and optimization

$4000

Please provide

  1. cell lines and culture medium
  2. antibody if needed

Library ( whole human genome siRNA Library)

$11000

  1. primary data analysis only
  2. Validation not included

Materials or reagent for screening

$5000

Multi-well plate, liposome 2000 (Invitrogen) or cell microarray

Salary

$30000

3 technician for 3 months

Total

$50000

 

 

 

 

Additional Library (miRNA Library)

$850

 

Additional Assay reagent (antibody et al)

$6000

 

 

 

 

 

Ordering
Download
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: 5OD, PAGE purified, $99/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, precise quantity in OD and nmols, Tm , MW, size, extinction coefficient and purification data.
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.