- What are the advantages and disadvantages of various RNAi methods?
1 Chemically-synthetic dsRNA: Advantage: fast, usually it only takes 6-8 days to receive custom RNA oligo from suppliers.
2 in Vitro Transcribed siRNAs: Advantages: relatively inexpensive. Disadvantages: operational difficulties and time-consuming.
3 siRNA expression cassettes (SECs) constructed by PCR: Advantages: economic and efficient. Disadvantages: PCR products are hard to be transfected into cells.
4 shRNA Expression Plasmid: Advantage: the gene suppression lasts longer; economic.
Disadvantage: Time-consuming; low transfection efficiency; It is also involved of the localization of plasmid inside cells, the in vivo shRNA folding efficiency and non-specific gene inhibition.
- What should be paid attention to when design siRNA?
SiRNA design needs to be accomplished by software, but there are some general rules:
1. The fragment is between the 75-100 nt of the start codon and the 100 nt of the stop codon;
2. The G/C ratio is between 35-50%, and it is best not to over 55%;
3 After the sequence design, BLAST homology analysis should be carried out. Attention: the choice of RNAi has certain randomness for any target gene.
- What should be used for dissolving RNA, water or buffer?
- We recommend using 1X TE buffer (10mM Tris-HCl, PH7.5; 0.1mM EDTA; RNase free) to dissolve single-stranded RNA. This solution can buffer the PH and chelate metal ions, then reduce the RNA degradation. You can also use RNase free water to dissolve the dsRNA freeze-dried from the 10 mM Tris-HCL, pH 8.0, 20 mM NaCl, 1 mM EDTA buffer, making a final RNA concentration of 20 μm.
- How to do the conversion between OD and nmol of RNA oligo?
There is a precise formula for the conversion between OD and nmol of RNA oligo, but in general, for a 21bp siRNA oligo, there is a simple calculation as follows:
1 OD duplex= 3 nmols = 40ug
- How to choose fit RNAi product among so many RNAi products?
- There are many RNAi products on the market, but they can roughly be divided into two categories: one is the chemically-synthetic RNA oligo; another one is the reagent kit based on molecular biology operation. Both of them are widely used in RNAi research. For the beginner of RNAi research, the better way is to synthesize a group of RNA oligo (4-5 duplexes) and screen the specific RNA oligo which can obviously down-regulate the expression of the target gene. Next, you can use vectors or relatively large number of RNA oligo according to your research needs. In general, if you want to observe the continued gene down-regulation to study gene function, the sable expression vector system will be better; and if you want to study the effects and results of a RNAi fragment as a drug, then synthesis of a large amount of RNA oligo will become necessary. Our GenePharma products will make your research convenient!
- How to detect the effect of RNAi?
- The most common method to detect specific gene suppression is the Western Blot, which is used to compare the change in the level of protein expression before and after siRNA delivery. In some cases, the report gene system, such as β-galactosidase, can be used to detect RNAi effect. You can also use RT-PCR or other kinds of analyses based on cells to detect the level of transcripts in cells.
- What are the name, absorption wavelength, emission wavelength and color in visible light of each fluorogenic dye we used?
- How do I calculate the amount of buffer needed to resuspend siRNA if I need a stock concentration of 20 uM?
siRNA from GenePharma is supplied as a dried pellet in nmol quantities.
Stock concentrations are calculated as follows:
(quantity of siRNA, nmol)/(volume for resuspension, uL) = stock concentration, umol/L.
Therefore, solve for the unknown and include unit conversions to make sure units cancel.
Example: You purchased 20 nmols of siRNA and would like a 50 uM stock solution
a. equation: (20 nmol)/ ? uL = 50 umol/L
b. solve for unknown: ? uL = (20 nmol)(1 L/50 umol)
c. unit conversions: ? uL = (20 nmol)(1 L/50 umol)(1 umol/1000 nmol)(10exp6 uL/1 L)
d. answer: ? uL = 400 uL
Therefore, use 400 uL of 1X RNase-free buffer to resuspend 20 nmol of siRNA to make a 50 uM solution.
- How do I convert between nmol and ug of siRNA?
Use the molecular weight of siRNA to convert between nmol and ug. If the specific molecular weight is not known, you may use the average molecular weight of siRNA, which
is 13,300 g/mol.
If, for example, you have 5 nmol of siRNA, the conversion would be performed using the following steps:
a. equation: ? ug = (5 nmol)(13,300 g/mol)
b. unit conversions: ? ug = (5 nmol)(13,300 g/mol)(mol/10exp9 nmol)(10exp6 ug/g)
c. answer: ? ug = 66.5 ug
Therefore, a 5 nmol quantity of siRNA is 66.5 ug.
- What quantity of siRNA is needed to conduct an in vitro experiment?
We recommend that a screening concentration of 100 nM* final siRNA concentration be used for initial experiments. Therefore, 1 nmol of siRNA is sufficient for preparation of 10 mL of a solution. This is generally enough volume to prepare one complete 96- or 24-well plate.
* 100 nM = 100 nmol/L = 100 pmol/mL = 100 fmol/uL