GE Healthcare

Protocol
Dharmacon siRNA Resuspension
This protocol is for the resuspension of synthetic RNAi reagents
Note: This protocol is written for siRNA, but may also be applied to microRNA mimic and hairpin inhibitor resuspension.
For detailed recommendations for resuspension of siRNA/microRNA in plates, please see our siRNA Library Guidelines.

1. Briefly centrifuge tubes containing siRNA to ensure that the siRNA pellet Table 1. Recommended siRNA resuspension
is collected at the bottom of the tube.
2. Resuspend in RNase-free 1x siRNA Buffer (See note below) for the
volumes and concentrations.
desired final concentration using volumes listed in Table 1. 1x siRNA Buffer to be added (L) for
a. For example: for 10 nmol of siRNA and a 20 M stock concentration, siRNA Amount desired final concentration
add 500 L 1x siRNA Buffer. (nmol)
100 M Stock 20 M Stock
3. Pipette the solution up and down 3-5 times, avoiding the introduction of
bubbles and securely seal tubes (or multi-well plates). 1.0 10 50
4. Place the solution on an orbital mixer/shaker for 30 minutes at room
temperature. 2.0 20 100
5. Briefly centrifuge tubes containing siRNA to ensure that the solution is
5.0 50 250
collected at the bottom of the tube.
6. Verify the concentration of siRNA using UV spectrophotometry at 260 10 100 500
nm. For siRNA, 1 M = 13.3 ng/L. For microRNA mimic, 1 M=14.1 ng/L,
and microRNA hairpin inhibitor, 1 M=18.5 ng/l see FAQs for additional 20 200 1000
information.
50 500
7. RNA may be used immediately, or aliquoted into smaller volumes to limit Exceeds tube volume
the number of freeze-thaw cycles. Resuspended siRNA should be stored 100 1000
at -20 C in a manual defrost or non-cycling freezer. Storage at 4 C is
suitable for up to 6 weeks.

Notes:
Synthetic RNAi reagents should be resuspended in RNase-free solutions. We recommend 1x siRNA Buffer (diluted from Dharmacon 5x siRNA Buffer,
Cat #B-002000-UB-100). For short-term storage, RNase-free water (Cat # B-003000-WB-100) is also appropriate for resuspension of concentrated
stocks.
Salts present in buffer are known to affect the absorbance reading of RNA. For the most precise readings, dissolve in 4 volumes of sterile RNase-
free water for spectrophotometric analysis. Then adjust with addition of 5x siRNA Buffer appropriately to desired final concentration of siRNA
adjusting to 1x siRNA Buffer. Please see the FAQ section on page 2.
To dilute the 5x siRNA Buffer to 1x siRNA Buffer, mix four volumes of sterile RNase-free water with one volume of 5x siRNA Buffer. The composition
of the 1x siRNA Buffer is 60 mM KCl, 6 mM HEPES-pH 7.5, and 0.2 mM MgCl2.
5x siRNA Buffer is not intended for in vivo applications, as it has not been optimized for physiological conditions. Instead, an appropriately buffered
RNase-free solution should be used.

Technical Considerations:
For efficient Dharmacon siGENOME siRNA, ON-TARGETplus siRNA, or miRIDIAN microRNA Mimic and Hairpin Inhibitor delivery, we strongly
recommend following the instructions provided by the manufacturer for the delivery method of choice (such as transfection reagent, or electroporation)
and taking measures to test and optimize the conditions best suited for the cell line or culture selected. For protocols using Dharmacon DharmaFECT
transfection reagents or Accell siRNA delivery, click here.
Assays for mRNA level, protein level, or phenotypic change may be performed to assess silencing effects. Because RNAi is an mRNA-specific event, we
highly recommend assaying for reduction at the mRNA level using reverse transcription quantitative real-time PCR (RT-qPCR). Typical time points for
detecting target knockdown with lipid-mediated siRNA or microRNA mimic delivery are 24-48 hours for mRNA and 48-96 hours for protein. Accell siRNA
delivery is typically assessed at 72 hours or longer. Time course studies are recommended to identify optimal time points for assessing knockdown.
Frequently Asked Questions:
Question Answer

RNA is most accurately quantified by measuring its absorbance at 260 nm (A260) with a dual beam
How do I quantitate the resuspended siRNA?
spectrophotometer.

Use Beers Law, A260 = ()(C)(L) where is the extinction coefficient (from the Product Transfer Form), C is
How do I calculate the concentration of the
the siRNA concentration, and L is the path length of the cuvette. Calculate the final concentration of the
siRNA sample?
resuspended siRNA by solving for C and multiplying by the dilution factor.

Salts present in 1x siRNA Buffer (or other resuspension solution) are known to cause a decrease in the
absorbance reading of RNA.
Differences in instrumentation for quantifying RNA may lead to differences in apparent values.
Dual beam UV-VIS spectrophotometers are recommended.
Why does the calculated amount of RNA
Sample is too concentrated. Absorbance values are most accurate between 0.15 and 0.6 and
in solution differ from that on the Product
within the linear range of a standard curve.
Transfer Form?
Sample is too diluted. Measurements with dilutions of small volumes (1-1.5 L) are more
susceptible to variation.
Sample may not be fully resuspended. Heat samples to 95 C for 1-3 minutes and allow to cool
for 30-45 minutes to reanneal complementary strands.

The siRNA has been at room

Yes. Samples are shipped as dried pellets and are stable at room temperature for 2-4 weeks. Upon receipt,
temperature for a week. Will the
we recommend that all samples should be stored at -20 C or -70 C to -80 C.
siRNA still be okay?

The average molecular weight (MW) of a siRNA is 13,300 g/mol.

What is the average molecular weight of
a siRNA, miRIDIAN Mimic , or miRIDIAN The average MW of a miRIDIAN mimic is 14,100 g/mol.
Hairpin Inhibitor?
The average MW of a miRIDIAN hairpin inhibitor is 18,500 g/mol

How do I convert between Multiply the number of moles by the MW on the Product Transfer Form, or the average MW for your oligo.
nmol to g of siRNA? For example, 5 nmol of siRNA would be: (5 nmol)(13,300 g/mol)(mol/109 nmol)(106 g/g) = 66.5 g

For additional Frequently Asked Questions (FAQs), please visit here.

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other trademarks are the property of General Electric Company or one of its subsidiaries. 2014 General Electric CompanyAll rights reserved. Version published June 2014.
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1.800.235.9880; 303.604.9499 if you have any questions.