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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
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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. Figure out 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. figure out the 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 Thus, use 400 uL of 1X RNase-free buffer to resuspend 20 nmol of siRNA to make a 50 uM solution.
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What is the name, absorption wavelength, emission wavelength and color in the visible light of each fluorogenic dye we used?
Abbreviation, Name, Absorption Wavelength, Emission Wavelength, Color: — 6-FAM 6-carboxy-fluorescein 494nm 518nm Green — TET 5-tetrachloro-fluorescein 521nm 538nm Orange — HEX 5-hexachloro-fluorescein 535nm 553nm Pink — TAMRA tetramethyl-6-carboxyrhodamine 560nm 582nm Rose — ROX 6-carboxy-x-rhodamine 587nm 607nm Red —Cy3 Indodicarbocyanine 552nm 570nm Red — Cy5 Indodicarbocyanine 643nm 667nm Violet.
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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.
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How to select the right product among so many RNAi products in the market?
There are many RNAi products on the market, but they can roughly be divided into two categories: chemically synthesized oligonucleotides; and reagent kits based on molecular biology operations. Both of them are widely used in RNAi research. For beginners of RNAi research, the better way is to synthesize a group of RNA oligos (4-5 duplexes) and screen the specific RNA oligos which can obviously down-regulate the expression of the target gene. Next, you can use vectors or a relatively large number of RNA oligos according to your research needs. In general, if you want to observe the continued gene down-regulation to study gene function, a stable 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 oligos will become necessary.
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How do I convert between OD and nmol for RNA oligonucleotides?
There is a precise formula for unit conversation. In general, for 21 bps siRNA and miRNA oligos: 1OD duplex = 2.5 nmol = 33ug, 1OD simplex = 5 nmol = 33ug
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What should I use 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.
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What should be paid attention to in siRNA design?
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 be over 55%; (3) After the sequence design, the BLAST homology analysis should be carried out. Note: the choice of RNAi has a certain randomness for any target gene.
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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 oligos from suppliers.
(2) in Vitro Transcribed siRNAs — Advantages: relatively inexpensive. Disadvantages: operationally complex and time-consuming.
(3) siRNA expression cassettes (SECs) constructed by PCR — Advantages: economic and efficient. Disadvantages: cells can be difficult to transfect with PCR products.
(4) shRNA Expression Plasmid — Advantage: gene suppression lasts longer; economic. Disadvantage: Time-consuming; low transfection efficiency; It also involves the localization of plasmid inside cells, the in vivo shRNA folding efficiency and non-specific gene inhibition.
