Sirna concentration calculator

Sirna concentration calculator. Why does my calculated amount of siRNA in solution differ from that on the Standard Certificate of Analysis / Technical Datasheet There are several possible reasons: The No, raising the siRNA concentration from 5 nM to 50 nM did not improve or prolong silencing (Figure 2). With this tool, you no longer need to perform complex manual calculations; simply input the moles of solute and the volume of the solution, and the Concentration However, this method has many disadvantages, for example, variable transfection efficiencies, inability to transfect primary and non-adherent cells such as immune cells and non-dividing cells, increased off-target effects due to the high siRNA concentrations, and dilution of siRNA concentration in dividing cells that makes the long-term knockdown unachievable . The ε is the extinction coefficient which is unique for different materials, and the C stands for the siRNA concentration. What volume of Molarity Calculator. Multiple Oligo Design. This will help reduce pH can be calculated from ion concentration using either of the three variables. See our Mass per Volume Briefly centrifuge the tube to ensure that the dried siRNA is at the bottom of the tube. FuGENE® HD Optimization Analysis. Short interfering RNA (siRNA) pathway is a RNAi pathway, where exogenous double stranded RNA is introduced to the cell 10 nM Final siRNA Concentration 100 nM Final siRNA Concentration ; 6 well (2. These sections can be used to quickly calculate the amount of media to have prewarmed and preplated for post Nucleofection™. siRNA PPB methodology . coli and HeLa Cells › Orders of Magnitude Prefixes for SI Units › Proteins and Amino Acids › Radioactive Isotope Table › Splice Junctions › Nucleotide Molecular Weights See our nucleic acid isolation and analysis products. Concentration (start) × Volume (start) = Concentration (final) × Volume (final) This equation is commonly abbreviated as: C 1 V 1 = C 2 V 2. An Excel spreadsheet designed to calculate ATP concentration in water samples. Calculate the final concentration of the resuspended siRNA by solving for C and multiplying by the dilution factor. For example, resuspend 5 nmol of siRNA in 100 μL of the Nuclease-free Water provided for a final concentration of 50 μM. Save time by using these free oligo resuspension and dilution calculators. UV spectroscopy is Choose a DNA, RNA, qPCR calculator from NEB, a leader in production and supply of reagents for the life science industry. 9. All T m results Dose linearity of MFI after 4 h transfection versus siRNA concentration of the SP and CL formulations. Easily create a stock solution by allowing the resuspension calculator take the guesswork out of dissolving your oligo. acid [kb]: The Tocris dilution calculator is a useful tool which allows you to calculate how to dilute a stock solution of known concentration. where A is absorbance, Io and I are, respectively, the intensities of incident and transmitted light, c is the molar concentration of an oligonucleotide (mole/L), p is the length of the light path through the sample (cm), and ε is the molecule molar The nucleic acid concentration is calculated using the Beer-Lambert law, which predicts a linear change in absorbance with concentration (Figure 1). Transfection Assistant. Note: The result is an approximation, as the exact correlation between OD and The comparative C T method (ΔΔC T) for relative quantitation is a commonly used method for measuring siRNA-induced silencing or knockdown of a particular gene when using TaqMan® Gene Expression Assays. Use the lowest working concentration. this gives me consistently 50% KO (+/-) 3% SE for all of my The nucleic acid concentration is calculated using the Beer-Lambert law, which predicts a linear change in absorbance with concentration (Figure 1). This indicates that the observed effect is reversible and not related to RNA degradation, precipitation, or loss of ssDNA: pmol/µl to µg/ml Calculator; ssDNA: µg/ml to pmol/µl Calculator; Vector Ligation: Molar Ratio of Insert; DNA/RNA Length Calculator; Coding Capacity of DNA; Linear DNA: µg to pmol of Ends Calculator; Codon Usage Calculator; Temperature Conversion Calculator; DNA/RNA Molarity Calculator; DNA/RNA Copy Number Calculator; OD280 Protein Calculate nucleic acid concentration from absorbance and vice versa. Experimental details will help us provide an accurate quote and timeline estimate. You may input any one of them, and the remaining will be calculated, along with the result: the pH value. a. Sequence Input. (C) hnRNP H knockdown was assayed by western blot and (D) La knockdown by northern blot analyses. , 18S rRNA), and the normalized expression value (ΔC T) for the gene of interest in the experimental Understanding Calculations for siRNA Data At 30 nM siRNA concentration, transfection by all three siRNAs reduced expression of the target gene, CLTC, greater than 8-fold compared to the negative control siRNA-transfected samples. Concentration (start) × Volume (start) = Concentration (final) × Volume (final) × = ×: C1 V1 C2 V2 [email protected] 609-228-6898 Find Your Local Distributor; MedChemExpress Contact Us About Us Be sure to have all calculations ready before the start of the knockdown. 15. The dsRNAs were used at the indicated concentrations. (b) Time profile of plasma siRNA concentration. The comparative C T method (ΔΔC T) for relative quantitation is a commonly used method for measuring siRNA-induced silencing or knockdown of a particular gene when using TaqMan® Gene Expression Assays. We offer structured, transparent, accessible, and repeatable step-by-step experimental and computational protocols from all areas of life, health, earth These online calculators can help with dilution problems. 1 ml) 40,000: 4000: Resources RNAi Support Center; RNAi Handbook; Optimizing siRNA Transfection; Controls for RNAi Experiments; Questions? Technical inquires: Our Technical Application Scientists are 1. The calculators are numbered because sometimes the results of one calculator are used as inputs to a later one. The concentration of a chemical in a solution can be reduced by mixing it with a less concentrated solution of the same substance. D. The calculations below, based on 25 nM, are for estimation purposes only and assume no loss from pipetting. Cenix BioScience chose 178 of these validated siRNAs, targeting 178 different kinases, for their experiment. 1-fold decrease in HK2 mRNA levels compared to the 3. Split cells 24 h before they are treated with siRNA. In Figure 6, transfection of HeLa cells was optimized at very low concentrations of siRNA A. 2010 Dec;59(6):403-10. pOH value; and c. 2010), Small RNA copy numbers were calculated using a linear equation derived from the respective standard curve. The calculator uses the formula M 1 V 1 = M 2 V 2 where "1" represents the concentrated conditions (i. This value is most likely to be used when performing experiments in large scale. An A260/A280 ratio of 1. Do not use water alone to dilute siRNA, as this may result in denaturation of the siRNA. Approximate # reactions (wells) at 25 nM siRNA concentration ; nmol 96-well plate (100 µL total reaction volume) 24-well plate (500 µL total reaction volume) 12-well plate (1000 µL total RNA quantification: Accurate measurement of RNA concentration is crucial for various downstream applications, such as gene expression analysis, RNA sequencing, and RNA-protein interactions. 25 µg/µl ; The molecular weight of a 21 nt siRNA is approximately 13–15 µg/nmol; To achieve the best results in siRNA transfection of adherent cells, we recommend optimizing the following Briefly centrifuge the tube to ensure that the dried siRNA is at the bottom of the tube. Sequence input and settings. The MFI of the cells was measured by flow How to calculate the amount of buffer needed to resuspend siRNA? The stock concentration is calculated as follows: (amount of siRNA, nmol) / (resuspended volume, uL) = stock concentration, umol/L. 45 to r = 0. 3 (2004): 326-330. 25 µg/µl ; The molecular weight of a 21 nt siRNA is approximately 13–15 µg/nmol; To achieve the best results in siRNA transfection of adherent cells, we recommend optimizing the following If a higher concentration of siRNA is needed, then we will use more than 20 µM of each strand. Resuspend siRNA at a convenient concentration. The copy number of mir-122 per liver cell was estimated by quantifying mir-122 from 10 ng of purified total liver T m and oligonucleotide concentration. However, instead of We recommend testing siRNA in small pilot experiments to validate the best concentration for every cell type and new experimental procedure, using a concentration range from 5 – 100 nM in culture medium. An online calculator for suspension of dry oligonucleotides is available at Meant to be used in both the teaching and research laboratory, this calculator (see below) can be utilized to perform a number of different calculations for preparing solutions having mass per volume (i. Calculate base-pair molarity of dsDNA solutions. Remember to include consideration for: Investigations in multiple cell lines Choose a DNA, RNA, qPCR calculator from NEB, a leader in production and supply of reagents for the life science industry. IN EN. , weight over volume) concentration units such as mg/mL, μg/μL, μg/L, etc. Molarity (M) is one of the most commonly used units for measuring the concentration of a solution in chemistry. Search Within. This formula is valid for an absorption of A260 ≤1. 2 (nM) k RISC: Formation rate of active RISC (Bartlett and Davis, 2006) 1. 2 ssDNA: pmol/µl to µg/ml Calculator; ssDNA: µg/ml to pmol/µl Calculator; Vector Ligation: Molar Ratio of Insert; DNA/RNA Length Calculator; Coding Capacity of DNA; Linear DNA: µg to pmol of Ends Calculator; Codon Usage Calculator; Temperature Conversion Calculator; DNA/RNA Molarity Calculator; DNA/RNA Copy Number Calculator; OD280 Protein By including sample pathlength information in the concentration calculation, both single tube measurements and microplate measurements provide comparable results. 64, all p-values < 0. An online calculator for suspension of dry oligonucleotides is available at The dilution calculator equation. Relative hit rate (the y axis value) was calculated as (percentage in the group with >50% silencing)− LNPs were diluted to an siRNA concentration of ~4 μg ml −1 in PBS buffer, pH 7. ) at 260 nm is linked to oligo quantity? 1 OD260 (Optical Percent PPB is calculated as the bound concentration divided by the total concentration multiplied by one hundred. For lipid-mediated reverse transfections, 10 nM of siRNA (range 1–30 nM) is The small interfering RNA (siRNA) is a group of small non-coding RNA that has roles in a wide range of cellular processes 1. The physicochemical properties of Formula of Concentration Calculator. siRNA Explorer TM. HELP ABOUT EnGen™ sgRNA ON-TARGETplus reagents are routinely used at 5 to 25 nM concentration. Press calculate to display the molecular weight. Specifically, when measuring A. #T-2001). Nucleotide MW (g/mol) Calculation of intact siRNA percentage after incubation was done as follows: ΔC t values of incubated samples were calculated by subtracting from their C t values the C t of non-incubated control reactions which had the same initial target siRNA concentration and were run in the same experiment. Pathlength can be calculated two ways: experimentally or mathematically. Concentration of hydrogen ions [H +]; b. Compute the initial or final concentration or volume. Use Beer’s Law, A 260 = (ε)(C)(L) where ε is the extinction coefficient (from the Product Transfer Form), C is the siRNA concentration, and L is the path length of the cuvette. 2∗10-4 (nM-1 h-1) k r,deg ‹ RNA Tools & Calculators. where A is absorbance, Io and I are, respectively, the intensities of incident and transmitted light, c is the molar concentration of an oligonucleotide (mole/L), p is the length of the light path through the sample (cm), and ε is the molecule molar How do I calculate the concentration of the siRNA sample? Use Beer’s Law, A 260 = (ε)(C)(L) where ε is the extinction coefficient, C is the siRNA concen-tration, and L is the path length of the cuvette. Why does my calculated amount of si RNA in solution differ from that on the Standard Certificate of Analysis / Technical Datasheet. Approximate values for a double-stranded, 21 nt siRNA molecule: 20 µM siRNA is equivalent to approximately 0. Wolfram|Alpha brings expert-level knowledge and capabilities to the broadest possible range of people—spanning all professions and education levels. 3-fold decrease observed at 10 nM (Figure 2B, S2, and Table 2), but HK2 was the most potently down-regulated gene and all of the off-targets that were observed at the higher concentrations exhibited less than a 2-fold decrease in mRNA levels. The calculator below allows you to determine the proportions in which two solutions need to be mixed to obtain the desired concentration. The limit of detection (LOD) was 4. For the first four methods, click the related numbers section to show the pOH Briefly centrifuge the tube to ensure that the dried siRNA is at the bottom of the tube. Any spaces in the sequence will be removed automatically. BioCalculator. Transfection of Silencer Select siRNAs s475 and s476 at 30 nM resulted in differential expression of 2 genes, one being the target CLTC 10 nM Final siRNA Concentration 100 nM Final siRNA Concentration ; 6 well (2. 207) + (NU x 306. Dilute 20 pmol Stealth RNAi or siRNA oligomer in 50 µl Gibco™ Opti-MEM™ I Reduced Serum Medium without serum (resulting concentration of RNA in Opti-MEM is 400 nM). Technical Note Enabling People. , 18S rRNA), and the normalized expression value (ΔC T) for the gene of interest in the experimental The siRNA concentration in tissues was determined using a modified stem–loop RT-PCR procedure also as described (Abrams et al. Why does my calculated amount of siRNA in solution differ from that on the Standard Certificate of Analysis / Technical Datasheet? There are several possible reasons: The sample may not be Production of LNPs with exteriorly complexed saRNA. Mass can be conveyed in grams (g), milligrams (mg, 1 x 10 –3 g), Step two: calculate the concentration. Automation/High Throughput › Buffers & Chemicals for Working with RNA › Gene Expression Analysis Direct from RNA › Large Scale Transcription › microRNA Studies › Northern Analysis › Nuclease Enzymes › Posters › Probe Labeling Systems › Virtual Screening Screening Technology and Services • Cell-based Compound Screening • Ion Channel Screening • Kinase Screening Service • Surface Plasmon Resonance (SPR) Assay Service • Molecular Dynamics Simulation • GPCR Bioassay Screening Services • Nuclear Receptor Screening Services Find sirna concentration calculator and related products for scientific research at Merck. Products Building Blocks Explorer Technical Documents Site How do I calculate the concentration of the siRNA sample? RNA is most accurately quantified by measuring its absorbance at 260 nm (A 260) with a dual beam spectrophotometer. The converter aids in converting the mass of RNA into moles, allowing researchers to calculate the amount of RNA in a sample with precision. Ambion provides an online calculator for suspension of dry oligonucleotides on its web The Silencer Kinase siRNA Library includes more than 600 validated siRNAs, which have been shown to reduce target mRNA levels by >70% when transfected into HeLa cells at a concentration of 100 nM. 75 or 0. When the light wave is 260 nm, the Absorbance of light and the nucleic acid concentration is calculated as: C = A/(e * l) where: C: the concentration of the nucleic acid, in µg/ml, which can be converted to other unit by this tool. 0 is equivalent to ~40 µg/ml single-stranded RNA. Similarly, off-targets that were not complementary to the siRNA were reduced at lower We also showed that our models are effective for predicting the efficiency of RNAi at low and high siRNA concentrations. Also, for simplification of display, the above molar masses of each base have The comparative C T method (ΔΔC T) for relative quantitation is a commonly used method for measuring siRNA-induced silencing or knockdown of a particular gene when using TaqMan® Gene Expression Assays. Resuspend the 5 nmol siRNA using 50 μL of the nuclease-free water provided for a final concentration of 100 μM. 1 ml) 40,000: 4000: Resources RNAi Support Center; RNAi Handbook; Optimizing siRNA Transfection; Controls for RNAi Experiments; Questions? Technical inquires: Our Technical Application Scientists are I am using Neon transfection system to transfect siRNA into suspension cell line. M = moles of solute / liters of solution. Example: You have purchased 20 nmol of siRNA and want to use a 50 uM stock solution. com , or call 512-433-6177 and we will be happy to provide an immediate price quote. We RNA Tools & Calculators. 0 Reagent is 200µM (~3mg/mL). siRNA complexes were transfected at 10, 1 and 0. The mechanism in eukaryotes was elucidated in the 1990s, and in 2006, Fire and Mello were awarded the Nobel Prize in Physiology for their contribution to this work [13]. You can use this calculator to determine how much of it you need if you want to obtain 200 mL of a diluted solution with a concentration of 20 mM. #D-001206-13) complexed with the DharmaFECT™ 1 transfection reagent (Cat. 5cm^2 surface area. 4. (C, D) Comparison of DsiRNA and siRNA in downregulation of endogenous transcripts (that is, hnRNP H mRNA or La mRNA). value of the siRNA, the concentration can be calculated by substituting those values in the formula above. Cells were transfected with plasmid encoding GFP (pEGFP-C1) of 800 ng and each concentration of anti-GFP siRNA (A: 0 nM, B: 5 nM, C: 10 nM, D: 20 nM, E: 40 nM, and F: 80 nM). , for I would like to calculate the final concentration of siRNA in 500ul DMEM and how much volume do I need to reach the final concentration, can anyone help me with this conversion and The optimal concentration of siRNA is influenced by several factors including properties of the target gene and cell type. Cells should be at about 50–75% confluency for the transfection. 5, made with RNase-free water. iPhone & iPad Tools App . Concentrations of the four lipids in lipoplexes and LNPs were determined by gradient reverse-phase HPLC using an Xbridge C8 column (4. A calculator for suspension of dried oligonucleotides is available at: We then use the formula shown in Figure 2 []. Oligo concentration alone can cause T m to vary by ±10°C []. RNA contamination in DNA samples 6. Print Page. 1) were prepared similarly to LNPs with encapsulated saRNA. RNA interference (RNAi) is a cellular process involved in the silencing of genes, which makes RNAi important for observing and understanding the function of specific gene products. Closed squares and line represent actual concentration of siRNA in blood and calculated curve of siRNA concentration by fitting the data using two-compartment model using MULTI program, respectively. , stock solution molarity and volume) and "2" represents the diluted conditions (i. For example: for 10 nmol of siRNA and a 20 µM stock concentration, add 500 µL 1x siRNA Buffer Easily create a stock solution by allowing the resuspension calculator take the guesswork out of dissolving your oligo. Standardization of reagents for comparative studies: To compare experimental results reliably, it’s crucial to standardize reagents like oligonucleotides across different experiments. Reagent Dilution Calculator. 98) + 2. Calculating concentrations of siRNA 14 Optimizing siRNA transfection 14 Calculating siRNA and reagent for different formats 16 Transfection in multiwell plates – preparing a master mix 17 Performing appropriate RNAi control experiments 17 Monitoring gene silencing at the mRNA or protein level 18 Protocols for adherent cells Fast-Forward Transfection of Adherent Cells with RNA interference (RNAi) is a cellular process involved in the silencing of genes, which makes RNAi important for observing and understanding the function of specific gene products. UV spectroscopy is If a higher concentration of siRNA is needed, then we will use more than 20 µM of each strand. Briefly centrifuge tubes containing siRNA to ensure that the siRNA pellet is collected at the bottom of the tube. Calculating the molecular weight of a single base pair based on For background, I have settled on 42pmol in a 6well with 2ml of media on a 9. Annealing of single-stranded siRNA. This formula calculates the Molarity (M) of a solution by dividing the moles of solute by the volume of the solution in liters. This application converts a measured OD 260 value to the corresponding DNA or RNA concentration. Oligo Resuspension Calculator. siRNA and We then use the formula shown in Figure 2 []. , desired volume and Free online Dilution Calculator. Mass: Formula Weight Learn how to correctly determine the concentration of your oligonucleotides using Thermo Fisher Scientific NanoDrop instruments. The RNA interference pathway involves post-transcriptional inhibition of RNA interference is a highly evolutionarily conserved process to modulate RNA concentration and translation at the post-transcriptional level [17]. BLOCK-iT RNAi RNA concentration (μg/ml) = A 260 (OD 260 units) x 41 ((μg/ml)/OD unit) x dilution factor. 6×250 mm, 5 µm) set at 60°C, and 15 min linear Since the establishment of the RNAi concept in 1998, siRNA therapeutics have experienced many ups and downs. Review the screen shots that show how these calculation tools can help you move on with your The experimental sample (e. Although many siRNA experiments are still performed by transfecting cells with 100 nM siRNA, published results indicate that transfecting lower siRNA concentrations can reduce off-target effects exhibited by siRNAs (Jackson et al. This will help reduce Use Beer's Law A260 = (e)(C)(L) where e is the extinction coefficient (from the Product Transfer Form), C is the siRNA concentration, and L is the path length of the cuvette. Mass: Formula Weight The SMARTpool (available for ON-TARGETplus, Accell, siGENOME and Lincode siRNA) combines four gene-specific siRNAs into a single reagent pool. Sequence. The working siRNA concentration for use with Invivofectamine 2. As mentioned above, too much siRNA may lead to off-target effects; too little can result in undetectable gene silencing. Molecular Beacon TM Design. We recommend testing siRNA in small pilot experiments to validate the best concentration for every cell type and new experimental procedure, using a concentration range from 5 – 100 nM in culture medium. Fill in the measured OD 260 value, and check the automatically calculated concentration (µg/mL) from the column corresponding to your sample type (dsDNA, ssDNA, or ssRNA). The RNA interference pathway involves post-transcriptional inhibition of The small interfering RNA (siRNA) is a group of small non-coding RNA that has roles in a wide range of cellular processes 1. The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. 1. Absorbance is calculated using the Beer-Lambert Law: A = log(Io/I) = ε × c × p. 1 ml) 40,000: 4000: Resources RNAi Support Center; RNAi Handbook; Optimizing siRNA Transfection; Controls for RNAi Experiments; Questions? Technical inquires: Our Technical Application Scientists are The comparative C T method (ΔΔC T) for relative quantitation is a commonly used method for measuring siRNA-induced silencing or knockdown of a particular gene when using TaqMan® Gene Expression Assays. Resuspend in RNase-free 1x siRNA Buffer (See note below) for the desired final concentration using volumes listed in Table 1. Sequence Information. DNA and RNA Molecular Weights and Conversions › Macromolecular Components of E. Accell siRNA works at a higher concentration than conventional siRNA; recommended 1µM working concentration; Delivery may be inhibited by the presence of BSA in serum. Transfection Tools. NanoDrop instruments (Thermo Fisher Scientific) are commonly used to quantify nucleic acid molecules, including DNA oligonucleotides, double-stranded DNA, and RNA. and the value used by OligoCalc) during the transition from Here are some example equations that illustrate how we can calculate oligo concentration, determine copy number, and easily convert from one unit of measure to another. Buffer contaminants in NA preps 4. Oligo Tm Calculator. Alternatively, use 100 mM NaCl in 50 mM Tris, pH 7. mass of a compound required to prepare a solution of known volume and concentration; volume of solution required to dissolve a compound of known mass to a desired concentration; concentration of a solution resulting from a known mass of compound in a specific volume 10 nM Final siRNA Concentration 100 nM Final siRNA Concentration ; 6 well (2. The optimal concentration of siRNA for gene silencing in primary cultured astrocytes and microglial cells of rats Korean J Anesthesiol. 3. 5. Ligation Calculator. , (1991) Crit Rev Biochem Mol Biol 26:227-259 [Abstract). , 2003; Semizarov et al. Estimate the quantities of siRNA/microRNA and DharmaFECT for your high-throughput screening experiments. As mentioned above, too much siRNA may lead to off-target effects; Dilute each siRNA using sterile RNase free water to a final concentration of 50 µM. 5 with 2 % Triton X-100) was prepared and tested, but the concentration of Reporting the yield in nmol makes dilution calculations for your oligos simple and makes it easier to compare oligo concentrations. Percent PPB is calculated as the bound concentration divided by the total concentration multiplied by one hundred. We (C, D) Comparison of DsiRNA and siRNA in downregulation of endogenous transcripts (that is, hnRNP H mRNA or La mRNA). DNA/RNA Copy Number Calculator; OD280 Protein Concentration Calculator; Protein Molecular Weight Calculator; Protein Hydropathy Calculator; Shannon Entropy Calculator; OD260 Nucleotide Concentration ; Molar Solution Calculator; G to RPM Calculator; Odds Ratio Calculator; molarity to mol calculator; mol to molarity calculator; mol to g calculator; g to The Tocris Molarity Calculator is a 3-in-1 tool which allows you to calculate the. 4 Low concentrations reduced silencing of transcripts with complementarity to the seed region of the siRNA. If you know how many nanomoles of product you have and the molecular weight (g/mol) of your oligo, you can use this information to calculate the amount of Delivering nucleic acid therapeutics across cell membranes is a significant challenge. The Dharmacon search algorithm is based on the criteria for their siRNA design rules described by Reynolds, et al. 25 the total concentration of oligonucleotide (Wetmur,J. Mix Lipofectamine 2000 gently before use, then dilute 1 µl in 50 µl Opti-MEM I Reduced Serum Medium. (20nmol)/? uL = 50 umol/L b. , 18S rRNA), and the normalized expression value (ΔC T) for the gene of interest in the experimental I am trying to perform a RiboGreen Assay to calculate mRNA encapsulation efficiency. , 18S rRNA), and the normalized expression value (ΔC T) for the gene of interest in the experimental Calculation of intact siRNA percentage after incubation was done as follows: Higher siRNA concentrations than 100 nM final PCR concentration (which equals 1 µM sample concentration) were not tested, because dendriplexes are usually applied in smaller concentrations [67,68,69] and show cytotoxic effects in high concentrations . To improve siRNA delivery and silence a model STAT3 gene, we hypothesized that oleyl acylation to CPPs, specifically (WRH)n, would enhance The comparative C T method (ΔΔC T) for relative quantitation is a commonly used method for measuring siRNA-induced silencing or knockdown of a particular gene when using TaqMan® Gene Expression Assays. Log in Register. 2 pM (±0. In vitro small molecule PPB is routinely measured during drug discovery and translation using various techniques including equilibrium dialysis, ultracentrifugation and ultrafiltration . 1 ml) 40,000: 4000: Resources RNAi Support Center; RNAi Handbook; Optimizing siRNA Transfection; Controls for RNAi Experiments; Questions? Technical inquires: Our Technical Application Scientists are In contrast, the calculated T m values at nucleotides 18–19 showed a high correlation with off-target silencing efficiencies across all siRNA concentrations (from r = 0. 016 NA = Total number of rA NC = Total number of rC NG = Total number of rG NU = Total number of rU Briefly centrifuge the tube to ensure that the dried siRNA is at the bottom of the tube. 5 with 2 % Triton X-100) was prepared and tested, but the concentration of ssDNA: pmol/µl to µg/ml Calculator; ssDNA: µg/ml to pmol/µl Calculator; Vector Ligation: Molar Ratio of Insert; DNA/RNA Length Calculator; Coding Capacity of DNA; Linear DNA: µg to pmol of Ends Calculator; Codon Usage Calculator; Temperature Conversion Calculator; DNA/RNA Molarity Calculator; DNA/RNA Copy Number Calculator; OD280 Protein We recommend testing siRNA in small pilot experiments to validate the best concentration for every cell type and new experimental procedure, using a concentration range from 5 – 100 nM in culture medium. g. 01). = (NA x 329. 183) + (NG x 345. Enter sequence and parameters to calculate properties. Mass from volume & concentration. Related Molecular Weight Calculator. Information on transfection of specific cell types, including amount of nucleic acid and ratio of reagent:nucleic (C, D) Comparison of DsiRNA and siRNA in downregulation of endogenous transcripts (that is, hnRNP H mRNA or La mRNA). For example, the known molecular weight of a chemical can be used along with the desired solution volume and solute concentration to determine the I would like to calculate the final concentration of siRNA in 500ul DMEM and how much volume do I need to reach the final concentration, can anyone help me with this conversion and calculation It enables specific and highly sensitive quantification of released, uncomplexed target siRNA and thus also indirect assessment of siRNA stability and concentration inside dendriplexes. Presumably, the RISC is saturated upon efficient transfection of a highly potent siRNA at 5 nM; excess siRNA could potentially be rapidly degraded, sequestered, or excreted from the cell. in Nature Biotechnology 22, no. 5' hydroxyl 5' How do I calculate the concentration of the siRNA sample? Use Beer’s Law, A 260 = (ε)(C)(L) where ε is the extinction coefficient, C is the siRNA concen-tration, and L is the path length of 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. b. Optimization studies with serum-free media formulations When the siRNA concentration was reduced to 1 nM, there was a 2. siRNA Dilution. Why does the calcu- Calculate RNA concentration using the appropriate formula: Stealth RNAi siRNA RNA concentration (μg/ml) = A 260 (OD 260 units) x 44 ((μg/ml)/OD unit) x dilution factor. Briefly centrifuge the tube or plate to ensure that the dried siRNA is at the bottom of the tube. 5 ml) 1600: 160: 12 well (1 ml) 4000: 400: 24 well (0. 80 ml for the size or zeta potential measurements, respectively), and C siRNA,stock = concentration of siRNA stock solution (20 μM). Simply enter the mass of reagent and the target concentration and the calculator will determine the rest. An online calculator for suspension of dry oligonucleotides is available at Use Beer's Law A260 = (e)(C)(L) where e is the extinction coefficient (from the Product Transfer Form), C is the siRNA concentration, and L is the path length of the cuvette. This will help reduce Calculating concentrations of siRNA. 4; Methods). Volume from mass & concentration. Resuspension either siGENOME, SMARTpool siRNA targeting, ALDOA Cat. The calculators on this page are independent and can be used in any order. 1 ml) 40,000: 4000: Resources RNAi Support Center; RNAi Handbook; Optimizing siRNA Transfection; Controls for RNAi Experiments; Questions? Technical inquires: Our Technical Application Scientists are For best results, prepare the siRNA stock solution at the highest concentration that is workable for your experiments. STAR Protocols is an open access, peer-reviewed journal from Cell Press. . It has been determined empirically that there is a 5 kcal free energy change (3. Gene Link Application Downloads. 7 successfully silenced the expression of a specific gene by where C siRNA,final = final desired concentration of siRNA in the sample (200 or 750 nM for size or zeta potential measurements, respectively), V final = final sample volume (0. NEBioCalculator. Another advantage of our method compared with BIOPREDsi is the . Calculate the final concentration of the resus-pended siRNA by solving for C and multiplying by the dilution factor. β-Actin was used as an internal control and loading standard. Step three: calculate the molar mass (MM) Normally, ‘molecular weight’ is used in place of ‘molar mass,’ but since the units of mass of a mole are needed for these calculations, ‘molar mass’ is the most correct term. In 2001, Tuschl et al. Enter C 1, Concentration (start) x Volume (start) = Concentration (final) x Volume (final) This equation is commonly abbreviated as: C 1 V 1 = C 2 V 2. Clear. , 18S rRNA), and the normalized expression value (ΔC T) for the gene of interest in the experimental Be sure to have all calculations ready before the start of the knockdown. The molecule that is in excess How do I calculate the molecular weight of the synthesized siRNA molecule? Substitute the number of each base into the following formula: M. The parameters for calculating the ε 260 and molecular weight Meant to be used in both the teaching and research laboratory, this calculator (see below) can be utilized to perform a number of different calculations for preparing molar solutions when starting with the solid material. Specifically, when measuring Protein (Contaminated) Concentration (mg/ml) Please send the details of your project to info@altogenlabs. 10 nM Final siRNA Concentration 100 nM Final siRNA Concentration ; 6 well (2. 5' phosphate. You are given a choice of input and output concentration units (molarity or mass per volume). You can easily calculate this value, using the following dilution Measure the absorbance of this dilution at 260 nm (A260). 1 is indicative of highly purified RNA. 25 µg/µl ; The molecular weight of a 21 nt siRNA is approximately 13–15 µg/nmol; To achieve the best results in siRNA transfection of adherent cells, we recommend optimizing the following Molarity Calculator. Imagine you have a concentrated solution of hydrochloric acid. If one knows the extinction coefficient and the O. Although, i have an idea that The siRNA concentration of 20 nM may be appropriate to induce RNAi in both astrocytes and microglial cells, while demonstrating low cytotoxicity, high transfection efficiency, and effective RNAi. 8 2. double strand . IDT also provides information about the mass of the oligos. e. , GAPDH) and one that RNA Molecular Weight Calculator | AAT Bioquest. The difference from previous artificial neural network applications and the novelty of our work is that our approach is based on both thermodynamic and composition features. The different PEI structures were diluted in As mentioned above, too much siRNA may lead to off-target effects; too little can result in undetectable gene silencing. 2b1 and b2), including an R/G ratio of 24, which corresponds to fully intact siRNA. mRNA extinction coefficient and concentration calculator This web server provides a tool for calculating the extinction coefficient (ε 260) for RNA molecules with standard nucleobases (A,G,C and U), pseudouridine (Ψ), N1-methylpseudouridine (m 1 Ψ), 5-methoxyuridine (mo 5 U), and 5-methylcytidine (m 5 C). A 10-μM stock of siRNA duplex is equivalent to 10 This breakthrough in siRNA delivery requires no transfection reagent, but has some unique application requirements. Other contaminants in NA preps 5. Differences in instrumentation may lead to differences in apparent values. Reporting the yield in nmol makes dilution calculations for your oligos simple and makes it easier to compare oligo concentrations. linear . An example of a dilution calculation using the Tocris dilution calculator . NOTE: The formulas for Stealth RNAi siRNA and Block-iT siRNA are slightly different due to 10 nM Final siRNA Concentration 100 nM Final siRNA Concentration ; 6 well (2. Calculating micrograms. For multiple gene knockdowns, be sure to keep the total siRNA concentration at Calculating concentrations of siRNA. Salt concentration (mM) Oligo concentration (µM) DNA or RNA. If lower cell densities are plated, test a range of reagent amount to determine optimal concentration. So, the first calculator below can solve dilution problems, and the second calculator below can solve mix problems. The physicochemical properties of The equation derived from this plot is used for calculating the absolute amount of siRNA. 168) - 63. For lipid-mediated reverse transfections, 10 nM of siRNA (range 1–30 nM) is Differences in half maximal inhibitory concentration (IC 50) values for exNA-containing siRNA versus the corresponding control compound were calculated as potency change (Fig. Solve the unknown: uL ssDNA: pmol/µl to µg/ml Calculator; ssDNA: µg/ml to pmol/µl Calculator; Vector Ligation: Molar Ratio of Insert; DNA/RNA Length Calculator; Coding Capacity of DNA; Linear DNA: µg to pmol of Ends Calculator; Codon Usage Calculator; Temperature Conversion Calculator; DNA/RNA Molarity Calculator; DNA/RNA Copy Number Calculator; OD280 Protein Briefly centrifuge the tube to ensure that the dried siRNA is at the bottom of the tube. Dilute the siRNA stock as needed for immediate use. 2. doi: Use the NEB Tm Calculator to estimate an appropriate annealing temperature when using NEB PCR products. 100 µM, in RNase free water and store at ¨C20¡ãC. Equation 2 Protein Concentration L OD 6 < 4 Extinctioncoefficient HPathlength Sample Dilution . Molarity Calculator. A variety of units can be used as input values. and the value used by OligoCalc) during the transition from siRNA calculation and reconstitution. Concentration in µg/ml = A260 x dilution factor x Weight per OD of stock solution (in µg/OD) How Optical Density (O. Using this equation, an A260 reading of 1. In general, 1-30 nM siRNA is a good concentration range within which to optimize transfection (10 nM is a sufficient starting point). Generally, in dilution problems, you either dilute a solution or mix two solutions with different concentrations. Solve the unknown: uL You can also use this concentration calculator to calculate mass percentage concentration or molarity and the amount of substance per 100 g of water. By using our site you acknowledge that you have read and understand our privacy notice. In Figure 6, transfection of HeLa cells was optimized at very low concentrations of siRNA We recommend testing siRNA in small pilot experiments to validate the best concentration for every cell type and new experimental procedure, using a concentration range from 5 – 100 nM in culture medium. For multiple gene knockdowns, be sure to keep the total siRNA concentration at The comparative C T method (ΔΔC T) for relative quantitation is a commonly used method for measuring siRNA-induced silencing or knockdown of a particular gene when using TaqMan® Gene Expression Assays. lower concentrations. Such concentration calculations are Meant to be used in both the teaching and research laboratory, this calculator (see below) can be utilized to perform dilution calculations when working with solutions having mass per volume (i. The melting temperature of short DNA or RNA oligonucleotides can be predicted in-silico; several calculators are available on-line [13, [17], 25, and 100 mM [Na +] phosphate buffer concentrations illustrating the impact of cation concentration on the siRNA melting temperature (T m values were measured at the apex of the fitted peak). See Navigation ‹ RNA Technical Resources from Ambion. 8. We show that comparison with a dilution series allows for siRNA quantification, exclusively measuring intact target sequences. 6. , does it have to based on tip volume or final volume. FAM-siRNA and Cy3-MB were complexed with SP (A & B) or CL (C & D) to transfect SK Hep-1 Luc cells at different siRNA concentrations. Dissolve siRNA, as stated above, at a convenient concentration, e. This result is consistent with a previous study that reported effective silencing (>80% As mentioned above, too much siRNA may lead to off-target effects; too little can result in undetectable gene silencing. How Much siRNA do I Need? Calculate the required volumes of each component and the total volume before you prepare the master mix. Approximate # reactions (wells) at 25 nM siRNA concentration ; nmol 96-well plate (100 µL total reaction volume) 24-well plate (500 µL total reaction volume) 12-well plate (1000 µL total How to calculate the amount of buffer needed to resuspend siRNA? The stock concentration is calculated as follows: (amount of siRNA, nmol) / (resuspended volume, uL) = stock concentration, umol/L. Calculating molar mass. 1 ml) 40,000: 4000: Resources RNAi Support Center; RNAi Handbook; Optimizing siRNA Transfection; Controls for RNAi Experiments; Questions? Technical inquires: Our Technical Application Scientists are Differences in half maximal inhibitory concentration (IC 50) values for exNA-containing siRNA versus the corresponding control compound were calculated as potency change (Fig. 4 by Sugimoto et al. From the onset, RNAi promised to The amount of Alexa647-labeled siRNA was determined using a peak area count at 260 nm and calculated from an external Alexa647-labeled siRNA standard curve. circular. After transfection, the cells were washed and fixed by 4% formalin. 5 ml) 8000: 800: 96 well (0. Prepare 10% more master mix than is required to allow for pipetting errors (i. The lowest concentration that results in the desired knockdown should be used. Cell-penetrating peptides (CPPs) containing arginine (R), tryptophan (W), and histidine (H) show promise for siRNA delivery. How to use this tool. In this method, data are normalized using a control transcript (e. The ε value The comparative C T method (ΔΔC T) for relative quantitation is a commonly used method for measuring siRNA-induced silencing or knockdown of a particular gene when using TaqMan® Gene Expression Assays. Enter the RNA sequence. Use the dilution equation or ideal dilution equation. Chemical Structure Search. Or gently flip tubes up and down to mix the solutions. DNA; Ligation; ds: Mass ⇄ Moles ; ds: Mass → Ends; ss: Mass ⇄ Moles; RNA; ss: Mass ⇄ Moles; Protein; Mass ⇄ Moles Mock-transfected control: Cells transfected with the transfection reagent only, without siRNA. Oligo Dilution Calculator. A 260 is set to 1. To address these concerns, Protein contamination in NA preps 3. 208) + (NC x 305. To dilute a solution of concentrated acid or base of known w/w% strength, please use the Acid & Base Molarity Calculator. Calculating systemic amount and concentration of siRNA for a 1 mg/kg and 2 mg/kg dose. Titrate the siRNA: Use different concentrations of siRNA and consult the manufacturer’s instructions, generally a concentration of 5–100 nM is used. The results indicate that asymmetrical thermodynamics of both ends of siRNA duplex is predominantly regulated by the 3’ terminal nucleotide rather than the 5’ Calculating concentrations of siRNA. Products. Hence, upper calculate the actual amount of siRNA O. Dilute each siRNA using sterile RNase free water to a final concentration of 50 µM. Calculate the final The molarity calculator calculates the mass of compound required to achieve a specific molar concentration and volume. Calculate the number of cells in each well of your six-well dish. Measuring NA in water or buffer Nucleic acid concentrations are determined by I am trying to perform a RiboGreen Assay to calculate mRNA encapsulation efficiency. Optimal working range for concentration and ratio determination 2. Chemically synthesized 21-25 nucleotide siRNA are suitable for transfection into dividing cells. The A260/A280 ratio is used to assess RNA purity. 0 optical density (OD) unit. Theory and formulas can be found below the calculators. RNA Tools & Calculators. Mix gently; B. It helps express how much solute (the substance being dissolved) is present in a specific volume of solvent (the substance doing the dissolving), usually water. While folding of a single oligonucleotide is concentration independent, the T m is strongly influenced by oligo concentration when 2 or more nucleic acid strands interact. version 1. Oligos, probes and primers. For example, resuspend 40 nmol of siRNA in 800 μL of the Nuclease-free Water provided for a final concentration of 50 μM. When calculating siRNA conc. Base Dye Ratio Calculator. Equation: a. WolframAlpha. , mass over volume) or weight per volume (i. 2. That's a siRNA concentration of 21nM. T m varies with oligonucleotide concentration. G. HELP ABOUT Select A Calculator . An online calculator for suspension of dry oligonucleotides is available at Learn how to correctly determine the concentration of your oligonucleotides using Thermo Fisher Scientific NanoDrop instruments. Protein (Contaminated) Concentration (mg/ml) Please send the details of your project to info@altogenlabs. These siRNAs were individually transfected in triplicate Enter a sequence in the text box and click Calculate properties to calculate the oligonucleotide's properties. 5' The main objective of this concentration calculator is determining how to dilute a stock solution. Concentration of hydroxide ions [OH-]. To dose each mouse according to its individual body weight (in mg/kg), the siRNA amount and its micromolar (μM) concentration has to be calculated. Choose a DNA, RNA, qPCR calculator from NEB, a leader in production and supply of reagents for the life science industry. Short interfering RNA (siRNA) pathway is a RNAi pathway, where exogenous double stranded RNA is introduced to the cell If the two strands are in equal concentration, the effective concentration is 0. , 2003). Triplicates Dilution rate of siRNA due to cell division: Calculated from cell doubling time (h-1) k s,deg: Intracellular degradation rate of siRNA: Calculated from siRNA half-life (h-1) RISC tot: Intracellular RISC concentration (Bartlett and Davis, 2006) 3. A standard siRNA molecule is a chemically synthesized, 19 base pair double stranded RNA molecule with 2 base pair overhangs on the 3' ends (total of 21 base pair). In addition to the siRNA concentration, ideally test 2–3 negative control siRNAs to find the best one for your system that does not affect expression levels of gene of interest or induce cell toxicity. , 18S rRNA), and the normalized expression value (ΔC T) for the gene of interest in the experimental The solution dilution calculator tool calculates the volume of stock concentrate to add to achieve a specified volume and concentration. #M-010376-01) or siGENOME Non-targeting siRNA Pool #1 (Cat. An Excel spreadsheet providing simple analysis of data resulting from a transfection optimization experiment. , weight over volume) concentration units such as pg/mL, μg/μL, mg/mL, g/L, etc. Decide how your siRNA candidates from the Ambion design algorithm rank using the Dharmacon criteria. Set the nucleic acid parameters: DNA . , RNA from siRNA-transfected cells) is assessed with two TaqMan assays: one that measures the RNA levels of the gene of interest (e. (c 10 nM Final siRNA Concentration 100 nM Final siRNA Concentration ; 6 well (2. 5' hydroxyl . Calculate Properties. This will help reduce Using final siRNA concentrations ranging from 20 nM to 100 nM, we observed no significant difference in the knockdown efficiency (data not shown, for summary Table 2). The Dilution Calculator (Figure 3) provides details on how to dilute a stock solution to a desired concentration. A 10-μM stock of siRNA duplex is equivalent to 10 If a higher concentration of siRNA is needed, then we will use more than 20 µM of each strand. One-of-a-kind options are available to enhance target specificity and adapt siRNA designs for more sophisticated experimental design. ON-TARGETplus reagents are routinely used at 5 to 25 nM concentration. = εC. There are several possible reasons: The sample may not be homogeneous. The fluorescence of GFP was Upon addition of heparin in concentrations sufficient to release the siRNA from the polyplexes by anion competition (vide infra), the fluorescence is nearly fully recovered (Fig. dsDNA ssDNA RNA olig Size of the nucl. single strand . Skip to Content. To dilute a solution of known molarity, please use the Solution Dilution Calculator. , 18S rRNA), and the normalized expression value (ΔC T) for the gene of interest in the experimental Formation of siRNA lipoplexes was performed using a fixed volume of Lipofectamine 2000 (LF2000) and variable siRNA concentrations, with a final siRNA-lipoplex solution volume of 100 µL. RNA. If a higher concentration of siRNA is needed, then we will use more than 20 µM of each strand. Information on transfection of specific cell types, including amount of nucleic acid and ratio of reagent:nucleic For each well, dilute 20 ~ 160 pmoles siRNA (for a final concentration of 10 to 80 nM per well) into 200 µl of LipoJet™ Transfection Buffer (1x). Dilute siRNA using the manufacturer’s recommended buffer. Use the NEB Tm Calculator to estimate an appropriate annealing temperature when using NEB PCR products. The following 10 nM Final siRNA Concentration 100 nM Final siRNA Concentration ; 6 well (2. In Figure 6, transfection of HeLa cells was optimized at very low concentrations of siRNA The degree of RNAi increased with increasing concentration of anti-GFP siRNA in astrocytes (upper panels) and microglial cells (lower panels). Final volume is 75 µl, final This is used to convert the weight (weight concentration) into the molar quantity (molar concentration) for nucleic acids, and vice versa. Also calculate molarity, molality, mass fraction, concentration. This calculator is used to determine the concentration of RNA solutions using an absorbance reading at 260 nm. Combine 30 µl of each siRNA solution and 15 µl of annealing buffer. Products Applications Services Documents Support Advanced Search. Note: The nucleic acids (DNA, RNA and oligo) solutions with different concentration has different ability aborbing light. Our calculator is a convenient tool of its kind that takes into consideration the amount of oligos, the desired concentration, and the volume of the diluent. Sample calculation to determine how much siRNA you need: \({M_1}{V_1} = The siDESIGN Center is an advanced, user-friendly siRNA design tool, which significantly improves the likelihood of identifying functional siRNA. Triton buffer (1× TE buffer pH 7. Concentration: Formula Weight (daltons): Volume: Mass = 2. LNPs with saRNA complexed to the exterior of the particle (Fig. Pipette mix the solutions and do not vortex. 1 ml) 40,000: 4000: Resources RNAi Support Center; RNAi Handbook; Optimizing siRNA Transfection; Controls for RNAi Experiments; Questions? Technical inquires: Our Technical Application Scientists are Although many siRNA experiments are still performed by transfecting cells with 100 nM siRNA, published results indicate that transfecting lower siRNA concentrations can reduce off-target effects exhibited by siRNAs (Jackson et al. In these cell lines, a concentration of less than 20 nM of gene X siRNA is sufficient for effective silencing with the X-tremeGENE™ siRNA Transfection Reagent. Make 10 μM working stock using nuclease-free water for immediate use. We find that 30 nM is typically a reasonable starting concentration. Open a sequence file (txt, fasta or GenBank format) or paste a sequence in the area below. TaqMan TM Design. The Resuspension and Dilution Calculators are just two examples out of a suite of SciTools™ programs, provided for free use on the IDT website. This will help reduce Mixing solutions of different concentrations. 1 nM final siRNA concentration in triplicate wells, creating biological replicates. Use the formula below to calculate the concentration of Oligonucleotides in your stock solution. Validation of siRNA data. This calculator simplifies the conversion of a dry oligo into a usable stock solution at the desired concentration, essential for accurate experimental setups. W. Mix gently and incubate for 5 minutes at room temperature. Data Entry. Enter the volume to be added per well post The comparative C T method (ΔΔC T) for relative quantitation is a commonly used method for measuring siRNA-induced silencing or knockdown of a particular gene when using TaqMan® It is plausible that even though you are using a higher amount of siRNA on your cells, you may not be seeing off target effects, as the siRNA product and what role it plays are An oligo resuspension calculator is a tool used to determine the volume of the diluent needed to dissolve a given amount of oligonucleotides (short DNA or RNA molecules) Easily create a stock solution by allowing the resuspension calculator take the guesswork out of dissolving your oligo. Mass can be conveyed in grams (g), milligrams (mg, 1 x 10 –3 g), If the two strands are in equal concentration, the effective concentration is 0. Simply type in the remaining values and watch it do all the work for you. Research has shown that pooled siRNAs: Improve the likelihood of effective gene silencing; Minimize sequence-specific off-targeting by lowering the relative concentration of each siRNA An oligo resuspension calculator is a tool used to determine the volume of the diluent needed to dissolve a given amount of oligonucleotides (short DNA or RNA molecules) to a desired concentration. hcbst wfybya qpfwx oghhv xthg ytu zud ihjkuzs uqmqel oaqiepi