Comprehensive guidance for ordering, handling, and conducting experiments with GeneMedi's siRNA products
siRNAs are short, noncoding, double-stranded RNAs that leverage the RNA interference (RNAi) pathway to achieve highly specific gene silencing. They suppress gene expression by targeting and selectively degrading complementary messenger RNA (mRNA), through RNA-induced silencing complex (RISC).
You may order siRNAs through our website. The desired target can be searched by Symbol, Gene No, accession Number. Each query will yield three candidates. You may select the desired quantity at that point.
You may order siRNAs for the target gene of your choice by entering the gene name or Accession number at our website and selecting one of the listed siRNA candidates. You may also order custom designed siRNA if your model organism is not Human, Mouse or Rat. You can also order siRNAs by sequences, please email us.
For siRNAs, both the sense and antisense strands are synthesized at equimolar concentrations, then annealed and delivered in duplexed, lyophilized form. EPDC-treated water with every order. We recommend reconstituting to 100μM.
With a 2.5 nmol scale siRNA order, you can transfect ten 24-well plates at 20μM per transfection. For in vitro experiments, the standard small-scale purification method is OPC desalt. For in vivo experiments, HPLC purification is required. For in vivo experiments, HPLC purification is required.
To purify newly synthesized siRNA, we have several methods including OPC, HPLC and PAGE. We select a purification method depending on the end-use of the product:
We have the ability to synthesize chimeric RNA molecules that incorporate DNA bases. We can also incorporate 2'-O-Methyl and 2'-F into the RNA.
The following modifications are offered for our siRNAs:
Unless explicitly stated, the 5' and 3' ends are capped with -OH groups. Therefore, to order 5' phosphate-capped siRNAs, you must request for 5' phosphorylation modification.
The maximum length for a single-stranded RNA (ssRNA) molecule is 45 bases. We charge per base for this type of order. For siRNA molecules, the maximum length is 30 bases, which is offered at a flat price.
siRNAs can normally be kept stable at -20°C for over 1 year. The lyophilized form is especially stable and has a long shelf-life. Although solution-dissolved siRNAs can be stable, contamination of the reconstitution solution with RNase will degrade the product. Also, repeated freeze-thaw cycles accelerate the degradation process. Therefore, we recommend that after you receive the siRNA stock, you reconstitute and make several aliquots to avoid such freeze-thawing.
If you would like to keep your siRNA in solution, we recommend reconstituting with DEPC-treated water that we provide with your order to maximize stability.
Each synthesis report gives you a 'volume for 100pmol/μL' value that stands for the volume of DEPC-treated water you need to add to achieve a 100pmol/μL concentration. As an example, if the 'volume for 100pmol/μL' values for two siRNAs are 100, then you can reconstitute with 100μl DEPC-treated water, respectively, to reach a 100pmol/μL concentration.
In other words, the siRNA contained in each tube is:
100μL x 100pmol/μL = 10,000pmol = 10 nmol,
Although the final use will dictate the concentration of siRNA, we found that for average use, 100pmol/μL is ideal. That is why we provide you with DEPC-treated water volume for reconstitution to 100pmol/μL.
The standard concentration units for oligomers is given in M (mol/L), and the prefixes such as μ (micro), n (nano), p (pico) etc. describe the scope of the unit. The following prefixes are used not only for M, but for other measurement units such as length, mass etc.
1pmol/μL = 1x10-12 mol / 1x10-6 L= 1x10-6 mol/L= 1μmol/L= 1μM
Therefore, μM and pmol/μL are one and the same.
Normally, we will fulfill an order with a guaranteed nmol amount, and the synthesis report will also report the final amount in nmols. If you must have the ng amount to calculate for an experiment, you can convert from nmol to ng by using the formula below. We make it easy for you by giving you the molecular weight of the siRNA sequence in the report.
Molecular Weight (g) X mole (nmol) = Mass of siRNA (ng)
One of the most important factors in a siRNA experiment is the assessment of whether the siRNA gets delivered into the cell. GeneMedi offers positive controls that can easily indicate whether the siRNA is being delivered successfully.
For siRNA transfection, we recommend that the cell density be approximately 70%. (HEK293 cell, 6-well plate standard: 1.5 x 105 cells/well. We also strongly encourage user optimization of these figures)
We recommend a starting concentration of 20nM, but strongly advise empirical optimization for the cell line and conditions of your experiment.
This is a source of confusion for many people. To transfect a single well with 20 nM siRNA, where the transfection volume is 2 mL, you will need 40 pmol of siRNA. Adding 2 µL of a 20 µM (20 pmol/µL) stock siRNA solution to 125 µL will yield 2.5 nmol of siRNA.
Photobleaching may occur if the fluorescent dye modified siRNA is exposed to light for prolonged periods of time. Therefore, we store such siRNAs in brown tubes and store that in a dark place. Dyes such as Cy3 and Cy5 are prone to degradation in solution environments where the pH is over 9. Therefore, avoid basic conditions when storing Cy3 and Cy5 modified siRNAs.
Because each cell line will have different transfection efficiencies for every transfection agent, we recommend that you select the transfection reagent most suitable for your cell line.
The Negative Control siRNA is a non-targeting siRNA that has low sequence homology to all known Human, Rat and Mouse sequences. Therefore, it can be used as a convenient negative control for all Human, Rat and Mouse siRNA experiments. The Positive Control siRNAs (human) demonstrate high knockdown efficiency for the target gene. Additionally, positive control siRNAs are available for reporter systems such as GFP and Luciferase.
You can easily verify the transfection efficiency by transfecting your cells with NC-Cy3 and observing the cells with a fluorescence microscope. The NC-Cy3 can also be used as a test reagent to optimize the transfection concentrations of both the siRNA and the transfection reagent.
The siRNA knockdown efficiency can be verified through various techniques including qPCR, Western Blot.
Please verify the siRNA integrity with PAGE analysis. If the siRNA is intact, please verify the transfection efficiency. GeneMedi has a policy of supplying 3 free siRNAs if all 3 single-target-gene predesigned siRNAs have less than 70% knockdown efficiency, provided you supply us with the following information:
With the control:
NC: Negative Control,
PC: GAPDH/GFP/Luciferase Positive Control siRNA,
Cell transfection efficiency NC: Fluorescein labeled Negative Control
First, because not all siRNAs will knock-down the target gene with identical efficiency, you should try 2-3 different sequences to find the best siRNA. Second, to make sure that the knock-down affects downstream protein expression, mRNA levels should also be measured. Thirdly, verify the knock-down phenotype by using another siRNA designed for the same target gene and show that the same phenotype appears.
Custom siRNA orders can be made and delivered in 10 business days.
Standard products are delivered in 5 business days.
