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QUANTITATION OF NUCLEIC ACID CONCENTRATION

Three methods are generally used for quantitation of nucleic acids :-

i) Optical density measurements.

ii) Ethidium bromide - agarose plates.

iii) Agarose gel electrophoresis estimation with a known standard DNA

i) Optical density readings

Description

The absorbance of UV light at 260 nm wavelength by nucleic acids gives an estimate of concentration, assuming firstly that there are no protein or phenol contaminants in the solution and secondly, that the concentration of the nucleic acid is greater than 250 ng / ml. The ratio of readings taken at 260 nm and 280 nm wavelengths (both in the UV range) gives an indication of the purity of the nucleic acid.

An OD unit corresponds to the amount of nucleic acid in µg in a 1 ml volume using a 1 cm path length quartz cuvette that results in an OD260 reading of 1.

For DNA OD260 1 = 50 mm / ml,

For RNA OD260 1 = 40 mg / ml,

For single stranded oligonucleotides OD260 1 ~ 33 mg / ml

For oligonucleotides of known base sequence, the concentration of a solution can be calculated using the extinction coefficients of the bases :-

dGTP = 11.7 ml / mmole dATP = 15.4 ml / mmole

dCTP = 7.3 ml / mmole dTTP = 8.8 ml / mmole

For any given oligonucleotide, multiply the number of times each base is present by its extinction coefficient and add the resulting four numbers to get the extinction coefficient (E) of the entire molecule. The concentration is given by OD260 = E x concentration in µmoles. A more approximate estimate of concentration in µmoles is given by the total OD260 divided by 10 x the length of the oligonucleotide in bases.

The ratio of readings taken at 260 nm and 280 nm wavelengths indicates of the purity of the nucleic acid :-

For pure DNA OD260 : OD280 = 1.8

For pure RNA OD260 : OD280 = 2.0

Ratios less than these indicate contamination of the solutions with eg. protein, phenol or guanidinium and the estimates of concentration will be inaccurate. Some impurities which interfere with UV OD readings can be removed by extraction of the preparation with n-butanol.

The OD280 : OD260 ratios of the individual bases are as follows :-

dGTP = 0.66 dCTP = 0.98

dATP = 0.15 dTTP = 0.7 UTP = 0.38.

Reagents and Equipment

UV spectrophotometer.

Quartz cuvettes.

Pure sterile water.

Method

1 Blank the spectrophotometer with water (use quartz cuvettes if using ultraviolet light) Make sure the cuvette is clean, especially if estimating RNA concentration 2 Dilute a known volume of nucleic acid solution in water 3 Take readings at OD260 & OD280 4 Calculate concentration of original solution. If 5 ml of a DNA solution or 4 ml of an RNA solution are diluted in 1000 ml, the concentration in mg / ml will be 10x the OD260 reading

ii) Ethidium bromide - agarose plates

Description

This method uses the UV-induced fluorescence of ethidium bromide dye intercalated into the nucleic acid. The amount of fluorescence is proportional to the amount of nucleic acid present. Dye bound to DNA has a much stronger fluorescence in UV light than free dye. UV light at 254 nm is absorbed by the DNA and transmitted to the dye and UV at 302 nm and 366 nm is absorbed by the dye itself. The energy is re-emitted at 590 nm in the red-orange part of the visible spectrum. Very small quantities of nucleic acid can be detected this way ~ 5 ng and quantitated by comparison with a series of standards. It is also useful when the solution contains contaminants that prevent UV absorbance readings. This method is most accurate for double-stranded DNA and for RNA. The intercalation of ethidium bromide into single stranded oligonucleotides is often not strictly proportional to their mass.

Reagents and Equipment

Agarose

Sterile water

Ethidium bromide - 10 mg / ml in water

Sterile petri dish

Method

1 Melt 0.4 g of agarose in 40 ml of water by boiling briefly, cool to 60oC and add 4 ml of 10 mg / ml ethidium bromide solution 2 Pour into a petri dish and allow to harden at room temperature 3 Spot a known volume of the sample and a series of standards onto the plate - use DNA standards for DNA and RNA standards for RNA - and allow to stand for at least 30 minutes. The small molecular weight contaminants that inhibit UV absorbance readings may also enhance or quench the UV-induced fluorescence of ethidium. They will have time to diffuse away from the nucleic acid sample by allowing the plate to stand 4 Photograph the plate whilst trans-illuminated with UV light if a permanent record is required and / or if there are numerous samples to be quantitated - UV light is harmful

iii) Agarose gel electrophoresis estimation with a known standard RNA

Description

This is a variant of method ii), using UV-induced ethidium bromide-fluorescence from the test RNA and from a known amount of a RNA standard. It also allows the simultaneous assessment of the integrity of the nucleic acid. It can be used for either RNA or DNA

Reagents and Equipment

Mini-gel equipment

Agarose

Sterile water

MOPS electrophoresis buffer

Formaldehyde

Ethidium bromide - 10 mg / ml in water

Formaldehyde - formamide - glycerol - BPB loading buffer

Standard RNA This should either be serial dilutions of an RNA approximately the same size as the test RNA or eg an RNA ladder (Promega) in which the amount of RNA in each sized fragment is known and one of the fragment sizes corresponds to the test RNA. A DNA standard can be used but quantitation will be less accurate

Method

1 Make a formaldehyde-ethidium-agarose mini-gel - see m7 2 Mix a known volume of sample with RNA loading buffer 3 Mix a known quantity of standard RNA with RNA loading buffer 4 Run the gel until the BPB dye front 2/3 of the way down the gel 5 Photograph the gel on a UV transilluminator and estimate the quantity of RNA in each sample by comparing the intensity of fluorescence with the known standards.

Reference

Reference #151 Tan Lab Library 07-94> Davis LG, Dibner MD, Battey JF. 1986 Basic Methods in Molecular Biology. Elsevier. New York

FootnortesFootnotes

1 Large amounts of tissue are used, proteins etc block the interface between the two GIT-CsCl and CsCl gradients, which traps RNA and significantly reduces the yield. Also, protein and DNA pass straight through the CsCl cushion and contaminate the RNA pellet 1 Addition of CsCl to the GIT lysate allows a gradient to be established in the tissue lysate during ultracentrifugation. This helps prevent the interface from becoming blocked by cellular debris and increases the yield by up to 5 fold 1 If the RNA yield is expected to be high, the rotor can be stopped after 16 - 18 hours 1 High concentration CsCl precipitates out at <14oC when spun at 180 000 g 1 RNA partitions into the aquoeus phase and DNA partitions into the phenolic phase when the pH < 8.0. Water saturated phenol has a pH of ~ 4.0 1 LiCl-RNA salts are insoluble in ethanol / isopropanol, whilst LiCl-DNA salts are relatively soluble. RNA from spleen and thymus particularly can become DNA contaminated and steps (5) and (6) reduce the amount of contamination 1 Addition of CsCl to the GIT lysate allows a gradient to be established in the tissue lysate during ultracentrifugation. This helps prevent the interface from becoming blocked by cellular debris and increases the yield by up to 5 fold 1 If the RNA yield is expected to be high, the rotor can be stopped after 6 hours 1 High concentration CsCl precipitates out at <14oC when spun at 180 000 g 1 RNA partitions into the aquoeus phase and DNA partitions into the phenolic phase when the pH < 8.0. Water saturated phenol has a pH of ~ 4.0 1 LiCl-RNA salts are insoluble in ethanol / isopropanol, whilst LiCl-DNA salts are relatively soluble. RNA from spleen and thymus particularly can become DNA contaminated and steps (4) and (5) reduce the amount of contamination 1 Phenol and GIT are miscible. Chloroform must be added to searate the two phases. Heating the GIT-phenol solution to 65oC increases the efficiency of the organic solvent extraction steps 1 Addition of CsCl to the GIT lysate allows a gradient to be established in the tissue lysate during ultracentrifugation. This helps prevent the interface from becoming blocked by cellular debris and increases the yield by up to 5 fold 1 If the RNA yield is expected to be high, the rotor can be stopped after 16 - 18 hours 1 High concentration CsCl precipitates out at <14oC when spun at 180 000 g 1 RNA partitions into the aquoeus phase and DNA partitions into the phenolic phase when the pH < 8.0. Water saturated phenol has a pH of ~ 4.0 1 LiCl-RNA salts are insoluble in ethanol / isopropanol, whilst LiCl-DNA salts are relatively soluble. RNA from spleen and thymus particularly can become DNA contaminated and steps (5) and (6) reduce the amount of contamination 1 Usually it is possible to use 5 ml of less GIT buffer 1 If small amounts of tissue are being harvested and the appropriate homogeniser probe is available, then use an Eppendorf tube and 0.5 - 1 ml of GIT buffer 1 Very small tissue fragments can be dissolved directly in 0.5 ml of GIT at 37oC 1 RNA partitions into the aqueous phase and DNA partitions into the phenolic phase when the pH < 8.0. Water saturated phenol has a pH of ~ 4.0 1 LiCl-RNA salts are insoluble in ethanol / isopropanol, whilst LiCl-DNA salts are relatively soluble. RNA from spleen and thymus particularly can become DNA contaminated and steps (5) and (6) reduce the amount of contamination 1 The pellet may be resuspended in GIT, and the phenol-chloroform extractions repeated a second or even third time. This is necessary for pancreas 1 Use 10 ml of lysis buffer per gram of tissue 1 Prolonged centrifugation makes the pellet very difficult to resuspend 1 RNA partitions into the aqueous phase and DNA partitions into the phenolic phase when the pH < 8.0. Water saturated phenol has a pH of ~ 4.0 1 LiCl-RNA salts are insoluble in ethanol / isopropanol, whilst LiCl-DNA salts are relatively soluble. RNA from spleen and thymus particularly can become DNA contaminated and steps (5) and (6) reduce the amount of contamination 1 The pellet may be resuspended in lithium-urea, and the phenol-chloroform extractions repeated a second or even third time. This is necessary for pancreas 1 mRNA constitutes approximately 1-5% of total RNA isolated from mammalian cells and 1g of oligo(dT) cellulose can bind up to 4 mg of poly(A+) RNA 1 LiCl-RNA salts are insoluble in ethanol / isopropanol, whilst LiCl-DNA salts are relatively soluble. RNA from spleen and thymus particularly can become DNA contaminated and steps (5) and (6) reduce the amount of contamination 1 mRNA constitutes approximately 1-5% of total RNA isolated from mammalian cells and 1g of oligo(dT) cellulose can bind up to 4 mg of poly(A+) RNA 1 LiCl-RNA salts are insoluble in ethanol / isopropanol, whilst LiCl-DNA salts are relatively soluble. RNA from spleen and thymus particularly can become DNA contaminated and steps (5) and (6) reduce the amount of contamination

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This page is maintained by Beverly Faulkner-Jones (b.jones@anatomy.unimelb.edu.au) using HTML Author. Last modified on 10/25/95.