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Inorganic phosphate determination as a method to quantify RNA
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 338 (2005) 165–167 www.elsevier.com/locate/yabio
Notes & Tips
Inorganic phosphate determination as a method to quantify RNA Elizabeth A. Runquist¤ Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA 94132, USA Received 14 October 2004 Available online 10 December 2004
Accurate quantitation of RNA is an essential tool in laboratories that are exploring RNA expression levels or making cDNA libraries. An accurate measure of template RNA provides the basis for precise probe/template, primer/probe/template, and primer/template ratios to ensure identical eYciency and linearity of reactions over a given range of RNA concentrations. It also provides the basis for comparing the concentrations of diVerent RNA preparations from the same or diVerent sample sources. Since probe/template and probe/primer/template interactions are stoichiometric, methods for RNA quantitation should provide a linear response with molar amounts of RNA. Methods to quantify RNA can be classiWed into two groups. The Wrst includes methods based on the absorption spectra of the purine and pyrimidine bases and the second includes a variety of Xuorescent dyes that bind RNA. The spectrometric method, which is used routinely by many laboratories, calculates RNA concentration based upon an average RNA nucleotide molar extinction coeYcient and an average molecular weight. Since this method relies upon the properties of a single average RNA nucleotide, it provides a linear response with molar quantities of RNA. While spectrometric measurements provide an estimate of RNA concentrations above 5 g/mL [1], there are inert errors associated with this method. First, there is some variability in both the absorption maximas and the molar extinction coeYcients of the four RNA bases [2]. Second, there is the dependence of the nucleotide spectra on pH and solvents. Third, at 260 nm, there is potential interference by protein, phenol, and thiocyanate [3].
*
Fax: +1 415 405 0540. E-mail address: [email protected].
0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.11.024
RNA quantiWcation using Xuorescent dyes such as ethidium bromide and RiboGreen minimizes the inert errors associated with the spectrometric methods and provides greater sensitivity. Ethidium bromide is useful for quantifying RNA concentrations greater than 200 ng/ mL [4], whereas RiboGreen can quantify very dilute RNA samples in the range of 1.0–50 ng/mL [5]. Although these Xuorescent dyes are extremely sensitive to RNA concentration and are for the most part insensitive to solvents, pH, and protein, the stoichiometric relationship between dye binding and RNA units (nucleotides) is not known. The lack of a linear relationship with molar amounts of RNA along with base selectivity [5] makes precise quantiWcation of RNA problematic. The purpose of this work was to develop a precise method for quantifying RNA that (1) provides a linear response with molar amounts of RNA, (2) is independent of RNA composition, (3) is insensitive to pH, protein, or solvents, and (4) has a sensitivity in the range of 0–3 g RNA. The basis for the RNA quantitation method described herein relies on quantifying inorganic phosphate. Described is a modiWcation of an inorganic phosphate assay developed by Duck-Chong [6] for determining phospholipid concentrations and the application of that assay to RNA quantiWcation. Escherichia coli 16-S and 23-S ribosomal RNA purchased from Roche Diagnostics (Indianapolis, IN) were diluted to 0.11 g/uL with diethylphosphoryl-cyanide-treated water and used as RNA standards. The phosphate standard was 0.5 mM Na2HPO4. Aliquots of standards were mixed with 6 l of 10% magnesium nitrate in methanol and heated to »95 °C until completely dry. Since determination of inorganic phosphate Wrst requires oxidization of the RNA phosphate esters to form orthophosphate, the following steps were performed in a hood. The dried samples were heated brieXy
166
Notes & Tips / Anal. Biochem. 338 (2005) 165–167
(15 s) in the upper Xame of a Bunsen burner (or Xame bottle) and then heated in the lower part of the Xame (tip of blue cone) until the sample turned white [6]. After cooling, 40 L of 6 N H2SO4 was added and the tubes were covered with marbles and heated at »95 °C for 15 min. Upon cooling, 0.2 mL of the colorimetric reagent—water/ammonia molybdate (2.5% w/v)/ascorbic acid (10%, w/v), 20:3:3 (by vol.)—was added, and then the tubes were heated for 5 min in a boiling water bath. The absorbance at 820 nm was measured in a microplate reader (Spec Max Pro). Several types of acids can be used to digest phosphate esters to inorganic phosphate including perchloric acid [7], hydrochloric acid [6], and sulfuric acid [8]. Flaming of the samples is required for both hydrochloric acid and sulfuric acid as it ensures complete phosphate oxidation. Initially we used perchloric acid using a modiWcation of the procedure described by Rouser et al. [7] and achieved results similar to those presented herein (data not shown) but due to safety issues we did not use this procedure routinely. Next we used the procedure by DuckChong [6], which utilizes hydrochloric acid at Wnal concentration of 0.83 N, but we were unable to achieve consistent results [9]. Finally, we found that sulfuric acid at a Wnal concentration of 1 N provided excellent results consistently in our microassay procedure. The use of sulfuric acid coupled with Xaming to ensure complete oxidation of the RNA phosphate assay detected 0–3.0 g of RNA (Fig. 1). To validate this method with that of the spectroscopic A260 method, the amount of inorganic phosphate of pure E. coli rRNA was experimentally determined using inorganic phosphate standards ranging from 0 to 10 nmol
Fig. 1. Representative standard curve for E. coli rRNA inorganic phosphate performed as described. Each point reXects the average of three samples.
and compared to the theoretical amount calculated from spectrometric A260 values. The theoretical amount of RNA phosphate was calculated using the conversion of 3.125 nmol P/g RNA based on the assumption that the average molecular weight of a RNA nucleotide is 320 and that there is one phosphate molecule per RNA nucleotide. An agreement of 88.9% (n D 20) was found. These results indicate that, like the spectrometric A260 method, the RNA inorganic phosphate assay provides linear response with molar amounts of RNA and can be utilized as an alternative method for quantiWcation. To verify that the RNA inorganic phosphate assay has a strict sensitivity toward phosphate, the sensitivity of the assay to protein and phenol contamination was examined and compared directly to that of the spectrometric A260 assay. The eVects of protein and phenol on the RNA inorganic phosphate assay were determined by adding either 24 g bovine serum albumin or 5 L of a 0.5% phenol solution with the RNA/magnesium nitrate aliquots (Fig. 2A).1 Likewise, the eVects of protein and phenol on the spectrometric A260 assay was determined by either adding 24-g bovine serum albumin or 5 L of a 0.5% phenol solution to varying amounts of E. coli rRNA in solution as described (Fig. 2B). As illustrated (Figs. 2A and B), the sensitivities of the RNA phosphate assay and the spectrometric A260 assay to RNA were virtually identical; however, the sensitivities of these two assays to the interfering substances were not. At the levels of protein and phenol used in this study, accurate RNA quantitation by the spectrometric A260 assay was not possible, whereas there was little eVect of protein and phenol on RNA quantitation by the phosphate assay. The RNA phosphate assay described here provides a method for RNA quantitation that is linear with molar amounts of RNA. This assay has a sensitivity range similar to that of the spectrometric A260 method (0–3 g RNA) but, given its strict speciWcity for phosphate, the RNA phosphate assay is not plagued by interference by protein, solvents, and pH or base speciWcity. Like the other assays available for RNA quantitation, the spectrometric A260 method and the Xuorescent assays that use either ethidium bromide or RiboGreen [4,5], the RNA phosphate assay is unable to discriminate between RNA and DNA The RNA phosphate assay should be useful for determining RNA concentrations on puriWed RNA preparations that are not contaminated with free nucleotides or DNA. With the inclusion of the RNA quantitation method described in this paper there are now three groups of methods available for determining amounts of RNA. In 1 This amount of protein in samples containing 3 g RNA resulted in an A260/280 ratio of about 1.5. This level reXects a considerable amount of protein contamination; most RNA isolation procedures A260/280 ratios above 1.6 to 1.8 (the ratio for pure RNA) are considered acceptable.
Notes & Tips / Anal. Biochem. 338 (2005) 165–167
167
requirement for sensitivity, which depends directly on the availability of the RNA samples. Basically, if the RNA sample amounts are extremely limited then the Xuorescent assays [4,5], which provide the greatest sensitivity, are required, and accuracy may be sacriWced unless standards of similar base length and composition are used. Second is the requirement for speed. Basically, if speed and cost are of greater concern than precision or sensitivity then the spectrometric A260 method is very simple and quick and requires simple basic laboratory equipment. This method provides an estimate of RNA concentration that is linear with molar amounts of RNA. Finally, if precise and consistent RNA concentrations are required for comparing multiple RNA preparations from multiple sources in which pH, base composition, protein contamination, and solvent interference may be variable then the RNA phosphate assay may be useful.
Acknowledgments We acknowledge the assistance provided by Mary Meredith in performing many of the assays and Jean Gustin for critically reading the manuscript. This research was supported by NIH MBRS-RISE Grant 5R25GM59298.
References
Fig. 2. Comparison of the RNA phosphate assay with the spectrometric RNA assay and eVects of protein and phenol on RNA determination. (A) The standard phosphate assay was performed using only rRNA (closed circles) or rRNA in the presence of protein (open triangles) or phenol (open circles). For samples containing protein or phenol, 24 g of bovine serum albumin or 5 L of a 0.5% phenol solution was added to the rRNA standards prior to drying the samples. (B). A spectrometric A260 assay was performed using only rRNA (closed circles) or rRNA in the presence of protein (open triangles) or phenol (open circles) diluted to 200 L with water. Protein, bovine serum albumin, was added to a Wnal concentration of 120 g /mL (the A260/ A280 ratio D 1.45 for 3 g RNA) and phenol was added to provide a Wnal concentration of 0.0125% (A260/A280 ratio D 1.46 for 0 g RNA).
selecting a method for determining concentrations of puriWed RNA there are three factors that should be considered: sensitivity, speed, and precision. First is the
[1] J. Sambrook, D. Russell, Molecular Cloning: A Laboratory Manual, vol. 3, third ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 2001, pp. A8.19–A8.20. [2] C.R. Cantor, P.R. Schimmel, Biophysical Chemistry Part II: Techniques for the Study of Biological Structure and Function, W.H. Freeman, San Francisco, CA, 1980 pp. 443. [3] C.R. Cantor, P.R. Schimmel, Biophysical Chemistry Part II: Techniques for the Study of Biological Structure and Function, W.H. Freeman, San Francisco, CA, 1980 pp. 381–385. [4] J.B. Le Pecq, C. Paoletti, A new Xuorometric method for RNA and DNA determination, Anal. Biochem. 17 (1966) 100–107. [5] L.J. Jones, S.T. Yue, C.Y. Cheung, V.L. Singer, RNA Quantitation by Xuorescence-based solution assay: ribogreen reagent characterization, Anal. Biochem. 265 (1998) 368–374. [6] C.G. Duck-Chong, A rapid sensitive method for determining phospholipid phosphorus involving digestion with magnesium nitrate, Lipids 14 (1978) 492–497. [7] G. Rouser, S. Fleischer, A. Yamamoto, Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots, Lipids 5 (1969) 494–497. [8] G.R. Bartlett, Phosphorous assay in column chromatography, J. Biol. Chem. 234 (1959) 466–468. [9] M. Meredith, E.A. Runquist, A Simple Cheap Sensitive Assay for Quantifying RNA, poster presented at ABRCMS, San Diego, CA, Oct. 2003.
Notes & Tips
Inorganic phosphate determination as a method to quantify RNA Elizabeth A. Runquist¤ Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA 94132, USA Received 14 October 2004 Available online 10 December 2004
Accurate quantitation of RNA is an essential tool in laboratories that are exploring RNA expression levels or making cDNA libraries. An accurate measure of template RNA provides the basis for precise probe/template, primer/probe/template, and primer/template ratios to ensure identical eYciency and linearity of reactions over a given range of RNA concentrations. It also provides the basis for comparing the concentrations of diVerent RNA preparations from the same or diVerent sample sources. Since probe/template and probe/primer/template interactions are stoichiometric, methods for RNA quantitation should provide a linear response with molar amounts of RNA. Methods to quantify RNA can be classiWed into two groups. The Wrst includes methods based on the absorption spectra of the purine and pyrimidine bases and the second includes a variety of Xuorescent dyes that bind RNA. The spectrometric method, which is used routinely by many laboratories, calculates RNA concentration based upon an average RNA nucleotide molar extinction coeYcient and an average molecular weight. Since this method relies upon the properties of a single average RNA nucleotide, it provides a linear response with molar quantities of RNA. While spectrometric measurements provide an estimate of RNA concentrations above 5 g/mL [1], there are inert errors associated with this method. First, there is some variability in both the absorption maximas and the molar extinction coeYcients of the four RNA bases [2]. Second, there is the dependence of the nucleotide spectra on pH and solvents. Third, at 260 nm, there is potential interference by protein, phenol, and thiocyanate [3].
*
Fax: +1 415 405 0540. E-mail address: [email protected].
0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.11.024
RNA quantiWcation using Xuorescent dyes such as ethidium bromide and RiboGreen minimizes the inert errors associated with the spectrometric methods and provides greater sensitivity. Ethidium bromide is useful for quantifying RNA concentrations greater than 200 ng/ mL [4], whereas RiboGreen can quantify very dilute RNA samples in the range of 1.0–50 ng/mL [5]. Although these Xuorescent dyes are extremely sensitive to RNA concentration and are for the most part insensitive to solvents, pH, and protein, the stoichiometric relationship between dye binding and RNA units (nucleotides) is not known. The lack of a linear relationship with molar amounts of RNA along with base selectivity [5] makes precise quantiWcation of RNA problematic. The purpose of this work was to develop a precise method for quantifying RNA that (1) provides a linear response with molar amounts of RNA, (2) is independent of RNA composition, (3) is insensitive to pH, protein, or solvents, and (4) has a sensitivity in the range of 0–3 g RNA. The basis for the RNA quantitation method described herein relies on quantifying inorganic phosphate. Described is a modiWcation of an inorganic phosphate assay developed by Duck-Chong [6] for determining phospholipid concentrations and the application of that assay to RNA quantiWcation. Escherichia coli 16-S and 23-S ribosomal RNA purchased from Roche Diagnostics (Indianapolis, IN) were diluted to 0.11 g/uL with diethylphosphoryl-cyanide-treated water and used as RNA standards. The phosphate standard was 0.5 mM Na2HPO4. Aliquots of standards were mixed with 6 l of 10% magnesium nitrate in methanol and heated to »95 °C until completely dry. Since determination of inorganic phosphate Wrst requires oxidization of the RNA phosphate esters to form orthophosphate, the following steps were performed in a hood. The dried samples were heated brieXy
166
Notes & Tips / Anal. Biochem. 338 (2005) 165–167
(15 s) in the upper Xame of a Bunsen burner (or Xame bottle) and then heated in the lower part of the Xame (tip of blue cone) until the sample turned white [6]. After cooling, 40 L of 6 N H2SO4 was added and the tubes were covered with marbles and heated at »95 °C for 15 min. Upon cooling, 0.2 mL of the colorimetric reagent—water/ammonia molybdate (2.5% w/v)/ascorbic acid (10%, w/v), 20:3:3 (by vol.)—was added, and then the tubes were heated for 5 min in a boiling water bath. The absorbance at 820 nm was measured in a microplate reader (Spec Max Pro). Several types of acids can be used to digest phosphate esters to inorganic phosphate including perchloric acid [7], hydrochloric acid [6], and sulfuric acid [8]. Flaming of the samples is required for both hydrochloric acid and sulfuric acid as it ensures complete phosphate oxidation. Initially we used perchloric acid using a modiWcation of the procedure described by Rouser et al. [7] and achieved results similar to those presented herein (data not shown) but due to safety issues we did not use this procedure routinely. Next we used the procedure by DuckChong [6], which utilizes hydrochloric acid at Wnal concentration of 0.83 N, but we were unable to achieve consistent results [9]. Finally, we found that sulfuric acid at a Wnal concentration of 1 N provided excellent results consistently in our microassay procedure. The use of sulfuric acid coupled with Xaming to ensure complete oxidation of the RNA phosphate assay detected 0–3.0 g of RNA (Fig. 1). To validate this method with that of the spectroscopic A260 method, the amount of inorganic phosphate of pure E. coli rRNA was experimentally determined using inorganic phosphate standards ranging from 0 to 10 nmol
Fig. 1. Representative standard curve for E. coli rRNA inorganic phosphate performed as described. Each point reXects the average of three samples.
and compared to the theoretical amount calculated from spectrometric A260 values. The theoretical amount of RNA phosphate was calculated using the conversion of 3.125 nmol P/g RNA based on the assumption that the average molecular weight of a RNA nucleotide is 320 and that there is one phosphate molecule per RNA nucleotide. An agreement of 88.9% (n D 20) was found. These results indicate that, like the spectrometric A260 method, the RNA inorganic phosphate assay provides linear response with molar amounts of RNA and can be utilized as an alternative method for quantiWcation. To verify that the RNA inorganic phosphate assay has a strict sensitivity toward phosphate, the sensitivity of the assay to protein and phenol contamination was examined and compared directly to that of the spectrometric A260 assay. The eVects of protein and phenol on the RNA inorganic phosphate assay were determined by adding either 24 g bovine serum albumin or 5 L of a 0.5% phenol solution with the RNA/magnesium nitrate aliquots (Fig. 2A).1 Likewise, the eVects of protein and phenol on the spectrometric A260 assay was determined by either adding 24-g bovine serum albumin or 5 L of a 0.5% phenol solution to varying amounts of E. coli rRNA in solution as described (Fig. 2B). As illustrated (Figs. 2A and B), the sensitivities of the RNA phosphate assay and the spectrometric A260 assay to RNA were virtually identical; however, the sensitivities of these two assays to the interfering substances were not. At the levels of protein and phenol used in this study, accurate RNA quantitation by the spectrometric A260 assay was not possible, whereas there was little eVect of protein and phenol on RNA quantitation by the phosphate assay. The RNA phosphate assay described here provides a method for RNA quantitation that is linear with molar amounts of RNA. This assay has a sensitivity range similar to that of the spectrometric A260 method (0–3 g RNA) but, given its strict speciWcity for phosphate, the RNA phosphate assay is not plagued by interference by protein, solvents, and pH or base speciWcity. Like the other assays available for RNA quantitation, the spectrometric A260 method and the Xuorescent assays that use either ethidium bromide or RiboGreen [4,5], the RNA phosphate assay is unable to discriminate between RNA and DNA The RNA phosphate assay should be useful for determining RNA concentrations on puriWed RNA preparations that are not contaminated with free nucleotides or DNA. With the inclusion of the RNA quantitation method described in this paper there are now three groups of methods available for determining amounts of RNA. In 1 This amount of protein in samples containing 3 g RNA resulted in an A260/280 ratio of about 1.5. This level reXects a considerable amount of protein contamination; most RNA isolation procedures A260/280 ratios above 1.6 to 1.8 (the ratio for pure RNA) are considered acceptable.
Notes & Tips / Anal. Biochem. 338 (2005) 165–167
167
requirement for sensitivity, which depends directly on the availability of the RNA samples. Basically, if the RNA sample amounts are extremely limited then the Xuorescent assays [4,5], which provide the greatest sensitivity, are required, and accuracy may be sacriWced unless standards of similar base length and composition are used. Second is the requirement for speed. Basically, if speed and cost are of greater concern than precision or sensitivity then the spectrometric A260 method is very simple and quick and requires simple basic laboratory equipment. This method provides an estimate of RNA concentration that is linear with molar amounts of RNA. Finally, if precise and consistent RNA concentrations are required for comparing multiple RNA preparations from multiple sources in which pH, base composition, protein contamination, and solvent interference may be variable then the RNA phosphate assay may be useful.
Acknowledgments We acknowledge the assistance provided by Mary Meredith in performing many of the assays and Jean Gustin for critically reading the manuscript. This research was supported by NIH MBRS-RISE Grant 5R25GM59298.
References
Fig. 2. Comparison of the RNA phosphate assay with the spectrometric RNA assay and eVects of protein and phenol on RNA determination. (A) The standard phosphate assay was performed using only rRNA (closed circles) or rRNA in the presence of protein (open triangles) or phenol (open circles). For samples containing protein or phenol, 24 g of bovine serum albumin or 5 L of a 0.5% phenol solution was added to the rRNA standards prior to drying the samples. (B). A spectrometric A260 assay was performed using only rRNA (closed circles) or rRNA in the presence of protein (open triangles) or phenol (open circles) diluted to 200 L with water. Protein, bovine serum albumin, was added to a Wnal concentration of 120 g /mL (the A260/ A280 ratio D 1.45 for 3 g RNA) and phenol was added to provide a Wnal concentration of 0.0125% (A260/A280 ratio D 1.46 for 0 g RNA).
selecting a method for determining concentrations of puriWed RNA there are three factors that should be considered: sensitivity, speed, and precision. First is the
[1] J. Sambrook, D. Russell, Molecular Cloning: A Laboratory Manual, vol. 3, third ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 2001, pp. A8.19–A8.20. [2] C.R. Cantor, P.R. Schimmel, Biophysical Chemistry Part II: Techniques for the Study of Biological Structure and Function, W.H. Freeman, San Francisco, CA, 1980 pp. 443. [3] C.R. Cantor, P.R. Schimmel, Biophysical Chemistry Part II: Techniques for the Study of Biological Structure and Function, W.H. Freeman, San Francisco, CA, 1980 pp. 381–385. [4] J.B. Le Pecq, C. Paoletti, A new Xuorometric method for RNA and DNA determination, Anal. Biochem. 17 (1966) 100–107. [5] L.J. Jones, S.T. Yue, C.Y. Cheung, V.L. Singer, RNA Quantitation by Xuorescence-based solution assay: ribogreen reagent characterization, Anal. Biochem. 265 (1998) 368–374. [6] C.G. Duck-Chong, A rapid sensitive method for determining phospholipid phosphorus involving digestion with magnesium nitrate, Lipids 14 (1978) 492–497. [7] G. Rouser, S. Fleischer, A. Yamamoto, Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots, Lipids 5 (1969) 494–497. [8] G.R. Bartlett, Phosphorous assay in column chromatography, J. Biol. Chem. 234 (1959) 466–468. [9] M. Meredith, E.A. Runquist, A Simple Cheap Sensitive Assay for Quantifying RNA, poster presented at ABRCMS, San Diego, CA, Oct. 2003.