Transcriptomics, the quantitation of RNA, is an import tool for research into gene function within biological systems.1 Techniques such as quantitative PCR2 and microarray3 enable the quantitation of RNA transcripts in tissues such as cartilage,4 bone,5 and muscle.6
Quality of RNA is a measure of RNA purity and integrity. The RNA integrity is of critical importance because downstream gene-expression profiling may be altered by changes in integrity.7,8 No gold-standard method exists to determine the quality of RNA extracted from tissue or cell cultures. Traditionally, visual assessment of an electrophoretic trace has been used as an identifier of RNA quality. Subsequently, the ribosomal band (28S:18S) ratio has also been used as an identifier of RNA quality, with a ratio > 2.0 indicative of high-quality RNA. A number of alternative metrics have been used to quantify RNA quality, such as the A260:A280 ratio,9 A260:A230 ratio, and ethidium bromide10 or SYBR green dye–stained agarose gel electrophoresis.
As low amounts of RNA are recovered from articular cartilage, quality assessment is ideally performed with the minimum amount of sample necessary. The recent development of microfluidic capillary electrophoresis has allowed the assessment of RNA quality11 with low volumes of sample (1 μL) through direct trace observation and automated calculation of the 28S:18S ratio. Analysis of RNA integrity can also be performed by use of computational software–based analysis of the electrophoretic trace.7,12 The DF12 is a figure calculated by use of a mathematic model examining degradation peak signals present in the lower molecular-weight range and by comparing them with ribosomal peak heights. A lower number denotes a higher-quality sample. The RIN is an algorithm that calculates RNA integrity from the electrophoresis trace by evaluating features such as the height of the 18S peak; the ratio of the area of the ribosomal bands, compared with the total area of the electropherogram; and the ratio of the fast area of the electropherogram to the total area of the electropherogram.13 A comparison of the RIN and DF of human tissue specimens reported that the RIN produces the most reliable data.7
Extraction and purification of RNA from articular cartilage are problematic. The tissue is relatively acellular, is usually available in only small quantities (with clinical canine samples often < 100 mg in wet weight), and contains a large amount of proteoglycan in the extracellular matrix.14 A variety of methods of RNA extraction from articular cartilage have been published to date. Most methods use liquid nitrogen and a dismembrator, phenol-chloroform RNA extraction and cesium trifluoroacetate ultracentrifugation,15 or silica membranea purification16 with or without17 isopropanol precipitation.
The purpose of the study reported here was to first investigate the benefit of isopropanol precipitation on the quality of RNA extracted from the articular cartilage from clinically normal dogs by use of different RNA quality metrics. Each RNA quality metric method was selected on the basis of the sample volume (ie, 1 μL) required to complete the test. We also assessed the relationship between RNA quality metrics to identify whether the results from 1 method infer those of another. Finally, a large number of RNA extractions from articular cartilage of clinically normal dogs and dogs with osteoarthritis were evaluated by use of low-volume RNA quality metrics to determine the value of quality information, compared with that of conventional quality assessment (ie, visual analysis of the electrophoretic trace). We hypothesized that the RNA quality metrics evaluating integrity could not be inferred from metrics evaluating purity (ie, absorbance ratios). We also hypothesized that RNA quality metrics evaluating integrity could be used to differentiate samples determined as being of high or low-quality RNA by visual analysis of an electrophoretic trace.
Materials and Methods
Articular cartilage from clinically normal dogs— Extraction of RNA was performed by use of methods similar to those described by Flannery et al17 and Reno et al.18 Articular cartilage from the femoral heads of 3 clinically normal crossbred dogs was harvested as previously described19 by sharp dissection and stored in a storage reagent.b Articular cartilage specimens were cut into 1-mm3 fragments and pooled. Aliquots of between 90 to 100 mg were dried, weighed, and snap-frozen in liquid nitrogen. Each aliquot was separately pulverized for 2 minutes at 2,000 oscillations/min in a liquid-nitrogen cooled dismembratorc and snap-frozen in a 1.5-mL centrifuge tube. A 1-mL aliquot of monophasic solution of phenol and guanidine isothiocyanate reagentd was added directly to the powdered cartilage, mixed, warmed to room temperature (approx 20°C), and allowed to stand for 30 minutes. Each sample was transferred to a 1.5-mL microcentrifuge tube and centrifuged at 12,000 X g for 10 minutes at 4°C. The supernatant was placed in a new 1.5-mL microcentrifuge tube, and the cell and tissue debris were discarded. Following the addition of 0.2 mL of chloroform, samples were vortexed for 15 seconds, allowed to sit at room temperature for 10 minutes, and then centrifuged at 12,000 X g for 15 minutes at 4°C. The upper aqueous phase was then removed. The RNA was purified by use of 1 of 2 methods. Once purified, RNA samples were stored at −80°C until analysis.
Method 1 of RNA purification
The upper aqueous phase was mixed with 0.5 mL of isopropanol and stored at −70°C overnight, followed by centrifugation for 10 minutes at 4°C and 12,000 X g, removal of the supernatant, and resuspension in 70% ethanol. The pellet was centrifuged at 7,500 X g for 5 minutes at 4°C. The 70% ethanol wash and centrifugation step was repeated, after which the supernatant was removed and the pellet resuspended in 30 μL of RNase-free water. Contaminating genomic DNA was then reduced by performing DNase digestion.e The RNA solution was then further cleaned by use of minicolumns and reagents according to the manufacturer's protocola and eluted in 30 μL of RNase-free water. The elution step was repeated with the elutant to maximize the amount of RNA eluted.
Method 2 of RNA purification
The upper aqueous phase was removed and mixed with an equal volume of 70% ethanol. The solution was then cleaned by use of minicolumns and reagents according to the manufacturer's protocol,a which included an on-column DNA digestion with an RNase-free DNase setf; final elution was performed in 30 μL of RNase-free water. The elution step was repeated with the elutant to maximize the amount of RNA eluted.
Articular cartilage from dogs with osteoarthritis— Extraction of RNA was performed by use of methods similar to those described by Flannery et al17 and Reno et al.18 Articular cartilage from the femoral heads of 37 dogs with osteoarthritis was obtained at surgery (ie, femoral head and neck excision or total hip arthroplasty) and immediately (< 15 minutes) placed in storage reagent.b Extraction of RNA from articular cartilage was performed on all the specimens from dogs with osteoarthritis by use of method 1 or 2. Once purified, RNA samples were stored at −80°C until analyzed.
Spectrophotometer measurements—Two UV spectrophotometers (Spec I and Spec II, respectively) were used to measure RNA quality. For measurement of RNA quality by use of Spec I,g 1 μL of each sample was dissolved in 39 μL of RNase-free sterile water and analyzed. The RNA concentration and Spec I A260:A280 ratio were recorded, and the concentration was adjusted to account for the dilution.
For measurement of RNA quality by use of Spec II,h 1 μL of each sample (undiluted) was evaluated. The RNA concentration, Spec II A260:A280 ratio, and Spec II A260:A230 ratio were recorded. All samples analyzed had an RNA concentration of > 20 ng/μL, as determined by use of Spec II.h
Electrophoresis—For each sample, 1 μL (undiluted) was analyzed.i The ribosomal band ratio (28S:18S), as calculated by use of a software programj from the area below each peak (ie, RR), was recorded. Further RNA quality metrics, DFs,12 and RIN,7 were calculated by use of available software programsj,k from the electrophoretic traces produced. Visual assessment of the electrophoresis traces was performed independently by 2 blinded observers experienced in analyzing RNA quality. Each sample was designated highor low-quality RNA. No descriptors were provided. For samples in which the 2 observers did not agree (n = 2), a third blinded observer assessed the trace and the sample was assigned a designation in accordance with the majority view.
Statistical analysis—The means, SDs, IQR, and 95% CI were calculated for each of the methods used, and different methods were compared by use of the t-test statistic, as a normal distribution was assumed because the values were obtained from the same tissue by use of the same method. Comparisons between tissues from unaffected (normal) and affected (osteoarthritic) joints were per formed with the Mann-Whitney U test because values from the same tissue extracted by different methods were pooled. Comparisons between samples of high- and low-quality RNA were performed with the Mann-Whitney U test. Correlations between different methods of RNA quality assessment were analyzed by use of Spearman correlation coefficients. Sensitivity and specific values were determined for each test by use of arbitrary cutoff values selected between the lower value of the 95% CI, for samples determined as being high quality by visual assessment, and the upper value of the 95% CI, and for samples determined as being low quality by visual assessment. Significance was determined at a value of P < 0.05. All data analyses were performed by use of a software program.1,m
Results
Articular cartilage from clinically normal dogs— No significant difference was identified between the quality of RNA extracted by the different methods (ie, Spec I A260:A280 ratio [P = 0.064]; Spec II A260:A280 ratio [P = 0.800]; Spec II A260:A230 ratio [P = 0.149]; RR [P = 0.507]; RIN [P = 0.681]; and DF [P = 0.872]; Figure 1). No significant correlations were identified between the variables measured (Table 1).
Spearman correlation coefficients for different metrics used to assess RNA quality of articular cartilage specimens from clinically normal dogs.
| Variables | |||||
|---|---|---|---|---|---|
| Spec I A260:A280 | Spec II A260:A280 | Spec II A260:A230 | RR | RIN | |
| Spec II A260:A280 | |||||
| Correlation coefficient | 0.543 | — | — | — | — |
| P value | 0.266 | — | — | — | — |
| Spec II A260:A230 | |||||
| Correlation coefficient | -0.580 | 0.348 | — | — | — |
| P value | 0.228 | 0.499 | — | — | — |
| RR | |||||
| Correlation coefficient | 0.169 | 0.338 | 0.051 | — | — |
| P value | 0.749 | 0.512 | 0.923 | — | — |
| RIN | |||||
| Correlation coefficient | 0.232 | -0.058 | -0.294 | 0.600 | — |
| P value | 0.658 | 0.913 | 0.572 | 0.208 | — |
| DF | |||||
| Correlation coefficient | -0.086 | -0.657 | -0.580 | -0.507 | -0.464 |
| P value | 0.872 | 0.156 | 0.228 | 0.305 | 0.354 |
— = Not applicable.
Quality metrics of 2 methods of RNA purification from articular cartilage specimens from clinically normal dogs. All measurements are in arbitrary units (ratios, RIN, or DF), and the values for each triplicate are displayed. M1 = Method 1. M2 = Method 2.
Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1438
Articular cartilage from dogs with osteoarthritis— Significant positive correlations were identified (Table 2) between the Spec II A260:A280 and Spec II A260:A230 ratios (P < 0.001), RR and Spec II A260:A280 ratio (P = 0.016), RIN and Spec I A260:A280 ratio (P = 0.001), RIN and Spec II A260:A280 ratio (P = 0.025), RIN and Spec II A260:A230 ratio (P = 0.017), and RIN and RR (P = 0.001). Significant negative correlations were identified between DF and Spec I A260:A280 ratio (P = 0.026), DF and RR (P < 0.001), and DF and RIN (P = 0.004).
Spearman correlation coefficients for different metrics used to assess RNA quality of articular cartilage specimens from dogs with osteoarthritis.
| Variables | Metrics used to assess RNA quality | ||||
|---|---|---|---|---|---|
| Spec I A260:A280 | Spec II A260:A280 | Spec II A260:A230 | RR | RIN | |
| Spec II A260:A280 | |||||
| Correlation coefficient | 0.389 | — | — | — | — |
| P value | 0.074 | — | — | — | — |
| Spec II A260:A230 | |||||
| Correlation coefficient | 0.240 | 0.745 | — | — | — |
| P value | 0.282 | < 0.001 | — | — | — |
| RR | |||||
| Correlation coefficient | 0.278 | 0.461 | 0.297 | — | — |
| P value | 0.211 | 0.016 | 0.132 | — | — |
| RIN | |||||
| Correlation coefficient | 0.730 | 0.487 | 0.515 | 0.669 | — |
| P value | 0.001 | 0.025 | 0.017 | 0.001 | — |
| DF | |||||
| Correlation coefficient | −0.523 | −0.171 | −0.124 | −0.681 | −0.761 |
| P value | 0.026 | 0.471 | 0.602 | < 0.001 | 0.004 |
See Table 1 for key.
Twelve samples were considered to be of low-quality RNA and 25 samples were considered to be of high-quality RNA by use of visual assessment of the electrophoretic trace. The Spec I A260:A280 ratio could not be calculated for 13 samples, 5 of which were of low quality and 8 of which were high quality. The DF could not be calculated for 7 of the samples, all of which were considered to be of low quality (as judged by the RIN and visual assessment of the electrophoretic trace), and an RIN could not be calculated for 8 samples, 6 of which were of high quality and 2 of which were low quality. The results were stratified into 2 groups (ie, low-and high-quality RNA; Figure 2). Values (median [IQR]) for each metric were as follows: Spec I A260:A280 ratio (low quality, 1.39 [0.98 to 1.70]; high quality, 1.87 [1.72 to 2.03]), Spec II A260:A280 ratio (low quality, 1.43 [1.02 to 1.82]; high quality, 1.91 [1.65 to 2.025]), Spec II A260:A230 ratio (low quality, 0.31 [0.11 to 0.58]; high quality, 0.68 [0.40 to 0.93]), RR (low quality, 0 [0 to 0]; high quality, 0.8 [0.65 to 1]), DF (low quality, 27.45 [12.92 to 34.17]; high quality, 5.91 [5.01 to 7.64]), and RIN (low quality, 1.1 [1.0 to 2.6]; high quality, 7.65 [7.0 to 8.0]). Significant differences were identified between all quality metrics (ie, Spec I A260:A280 ratio [P = 0.049], Spec II A260:A280 ratio [P = 0.021], Spec II A260:A230 ratio [P = 0.029], RR [P < 0.001], DF [P = 0.001], and RIN [P = 0.013]). Significant (P = 0.05) differences in quantity were also identified between samples of low (27.3 ng/μL [26.0 to 47.5 ng/μL]) and high quality (43.1 ng/μL [29.3 to 175.2 ng/μL]).
Box plots (panels A and B) of quality metrics of RNA purified from articular cartilage specimens from dogs with osteoarthritis. Box indicates IQR, line with in box indicates median, whiskers indicate 95% CI, and asterisks indicate outliers. Low and high quality were differentiated by visual assessment of the electropherogram. All measurements are in arbitrary units (ratios, RIN, or DF). Low = Low quality. High = High quality.
Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1438
Comparisons between groups—A significant difference in the quality of RNA from articular cartilage specimens from clinically normal dogs, compared with articular cartilage specimens from dogs with osteoarthritis, was identified by the Spec II A260:A230 ratio (P = 0.031) and RIN (P = 0.002) but not by the Spec I A260:A280 ratio (P = 0.275), Spec II A260:A280 ratio (P = 0.661), RR (P = 0.1876), and DF (P = 0.155). No significant (P = 0.982) difference in quantity was found between articular cartilage specimens from clinically normal dogs (median, 51 ng/μL; IQR, 26 to 68 ng/μL) and articular cartilage specimens from dogs with osteoarthritis (median, 35.1 ng/μL; IQR, 27 to 86 ng/μL).
RNA quality of articular cartilage from dogs with osteoarthritis—Six of 25 articular cartilage specimens from dogs with osteoarthritis determined as having highquality RNA could not have an RIN determined, yet all of these samples had a DF indicating high quality (ie, value < 10). Conversely, 7 of 12 articular cartilage specimens from dogs with osteoarthritis of lowquality RNA could not have a DF calculated, whereas in all samples, with the exception of 2 low-quality RNA samples, it was possible to ascribe an RIN value.
Sensitivity and specificity of RNA quality metrics—The sensitivity and specificity of each metric were compared with visual assessment of the electrophoretic trace (Table 3). The RIN and RR provided the most sensitive method for determining high-quality RNA, whereas the DF was most specific. The Spec I A260:A280, Spec II A260:A280, and Spec II A260:A230 ratios were found to be moderately informative with regard to RNA quality. By use of an algorithm, 36 of 37 samples could be ascribed to groups agreeing with the visual assessment of quality (Figure 3).
Sensitivity and specificity of different metrics for prediction of high-quality RNA, compared with visual assessment of the electrophoretic trace.
| Variables | Metrics used to assess RNA quality | |||||
|---|---|---|---|---|---|---|
| Spec I A260:A280 | Spec II A260:A280 | Spec II A260:A230 | RR | RIN | DF | |
| Value | 1.730 | 1.720 | 0.440 | 0.4 | 6.4 | 10.00 |
| Sensitivity | 81% | 72% | 72% | 92% | 100% | 88% |
| Specificity | 93% | 86% | 82% | 96% | 95% | 100% |
Proposed algorithm for determining high-quality (HQ) samples from low-quality (LQ) samples by use of DF, RIN, and RR.
Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1438
Discussion
Isopropanol precipitation of RNA allows separation of RNA from proteoglycans that partially copurify with RNA following phenol-guanidine thiocyanatechloroform extraction,20 and the isopropanol precipitation step essentially allows an extra cleaning procedure. However, our results indicate that there was no significant difference in RNA quality between the 2 methods for RNA purification of articular cartilage specimens from clinically normal dogs. On the basis of our results, there appears to be no benefit in performing the additional step of isopropanol precipitation during RNA extraction of articular cartilage.
Extraction of RNA from articular cartilage specimens of dogs with osteoarthritis was characteristically more degraded than that from unaffected articular cartilage, as determined by the RIN and Spec II A260:A230 ratio. A number of factors will contribute to these findings. Firstly, samples may not have been collected and stored consistently in the optimal manner because most were taken during a surgical procedure in which sample collection is not a priority. Secondly, markedly osteoarthritic tissue contains a proportion of cells that are apoptotic21 and thus likely to contain degraded or degrading RNA. Degradation of RNA has been observed as more rapid in canine tumor specimens, compared with unaffected tissue.22 Thirdly, articular cartilage specimens from clinically normal dogs were pooled, which would reduce the variability of the metrics assessed; thus, direct comparison may not be strictly valid. Finally, the RIN metric could not determine the RNA quality of 6 samples of high quality; thus, there is a bias in using this metric for assessment of articular cartilage specimens from dogs with osteoarthritis. A comparison of the methods of RNA purification on the quality of RNA purified from osteoarthritic articular cartilage tissues was not done, as no difference was found between the methods of extraction in unaffected (normal) cartilage specimens in the preliminary work, as well as the inherent variability of clinical sample quality.
No significant correlations were observed among RNA quality metrics with articular cartilage specimens from clinically normal dogs. This was probably a result of the small samples size (n = 6 values for each metric). Strong correlations were observed between the DF and the RIN and RR with articular cartilage specimens from dogs with osteoarthritis. This may have occurred because each of these values is generated from the same electrophoretic trace. The RIN had significant correlations with all other metrics, whereas the RR was significantly correlated with the Spec II A260:280 ratio and DF was weakly positively correlated with the Spec I A260:280 ratio. The reason for this was not clear but probably reflects the fact that the RIN could be calculated for most of the high-and low-quality RNA samples. A strong positive correlation was also found between the Spec II A260:280 and Spec II A260:230 ratios, probably because the same value for A260 is used in both calculations.
A major advantage of using cultured chondrocytes for expression analysis, rather than articular cartilage specimens from dogs with osteoarthritis, is that perfect-quality RNA (with no loss of integrity as determined by quality metrics) can be obtained. Monolayer cultured chondrocytes lack the large volume of extra-cellular matrix present in affected (osteoarthritic) cartilage specimens, most notably proteoglycans that are likely to interfere with RNA extraction profiles. Additionally, cell recovery can be performed directly into a solution for RNA recovery from the culture, without the requirement for pulverization. Although the culture of chondrocytes produces a “cleaner” system, their phenotype may become altered in culture.23 Comparisons of the level of gene expression between cell culture and in vitro samples have shown that although similar genes may be upregulated, the level of their expression can be widely different in comparison to evaluation of in vivo material.24
Quantity of RNA was not significantly different between articular cartilage specimens from clinically normal dogs and those from dogs with osteoarthritis. The quantities of RNA produced were all > 20 ng/μL, which, although < 50 ng/μL as recommended by the manufacturer for RNA quality assessment by electrophoresis, has been shown to produce RIN values and DFs that strongly correlate with visual assessment of quality.7 The quantity of RNA measured in affected (osteoarthritic) articular cartilage specimens of low quality was significantly lower than that measured in specimens of high quality. Quantity may contribute to the differences in RNA quality metrics identified between affected cartilage specimens of low and high quality. However, quantity should have no effect on the ultimate relative measurement of expression,25 whereas quality does7; thus, the determination of RNA quality metrics is still important.
An RIN and DF value could not be ascribed to 9 and 8, respectively, of the articular cartilage specimens from dogs with osteoarthritis. This is somewhat higher than previously published results assessing these tools7 and reflects the difficulty in obtaining RNA of sufficient quantity and quality in a clinical setting. For affected (osteoarthritic) articular cartilage specimens, RNA quality determination required calculation of RIN and DF values. Values of RIN were generated more consistently for low-quality samples, whereas DFs were generated more consistently for high-quality samples.
Although gene-expression profiles from partially degraded RNA samples have a high degree of similarity, compared with intact samples,26 results of other studies have shown that a significant decline can occur in the relative expression of genes in RNA samples of poor quality.7,8 With the advent of proposals for the minimum information about a microarray experiment,27 quality metrics of the original sample, such as capillary electrophoresis, can be added to control the quality of data produced.
Significant differences were observed among RNA quality metrics, when the articular cartilage specimens from dogs with osteoarthritis were stratified into high and low quality. There would be a tendency for visual assessment of the electrophoretic trace to select for integrity, rather than purity, because the trace generated by the analyzeri does not identify protein contamination unless it is bound to RNA. This is also true for agarose gel assessment of RNA with ethidium bromide. However, metrics assessing RNA purity did differentiate the samples when stratified by visual assessment of the electrophoretic trace, indicating that the tendencies for protein contamination and loss of integrity are linked.
On the basis of our results, we propose that the RR, RIN, and DF metrics are used to assess the quality of RNA obtained from canine cartilage. Although visual assessment of the electrophoretic trace allows a gross evaluation of RNA quality, use of graded metrics, such as the RIN, avoids individual error and provides a graded or scaled output. By use of the algorithm presented, 97% of articular cartilage specimens from dogs with osteoarthritis could be ascribed quality values that agreed with the visual analysis of the electrophoretic trace. Clearly, every effort should be made in the clinical setting to optimize articular cartilage specimen collection and storage to maximize specimen quality; however, some degree of integrity loss is to be expected, compared with cell culture systems. We recommend using multiple metrics for the accurate assessment of clinical articular cartilage specimens.
ABBREVIATIONS
| A260:A280 ratio | 260 to 280 nm absorbance ratio |
| A260:A230 ratio | 260 to 230 nm absorbance ratio |
| DF | Degradation factor |
| RIN | RNA integrity number |
| Spec I | First UV spectrophotometer |
| Spec II | Second UV spectrophotometer |
| RR | Ribosomal peak ratio |
| IQR | Interquartile ranges |
| CI | Confidence intervals |
RNeasy protocol for RNA, Qiagen Inc, Flemming Way, Crawley, UK.
RNAlater RNA stabilization reagent, Qiagen Inc, Flemming Way, Crawley, UK.
Braun Mikro-Dismembrator Vessel, B. Braun Biotech International GmbH, Melsungen, Germany.
Trizol solution, Invitrogen Corp, Paisley, UK.
RQ1 RNase-free DNase, Promega Corp, Madison, Wis.
RNase-free DNase set, Qiagen Inc, Flemming Way, Crawley, UK.
Ultrospec 2000, Pharmacia Biotech, Cambridge, UK.
NanoDrop ND-1000, Labtech International Ltd, Ringmer East Sussex, UK.
Agilent 2100 bioanalyser, Agilent Technologies UK Ltd, South Queensferry, West Lothian, UK.
Agilent Technologies, Palo Alto, Calif. Available at: www.chem.agilent.com. Accessed Mar 12, 2006.
Degradometer, version 1.41, Functional Genomics Core, Columbus, Ohio. Avaliable at: www.dnaarrays.org/downloads.php. Accessed Mar 12, 2006.
Minitab, version 14.1, Minitab Ltd, Coventry, UK.
SPSS 13.0 for Windows, SPSS UK Ltd., Woking, Surrey, UK.
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