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Letters to the Editor |
1 Department of Oncology-Pathology, Cancer Center Karolinska, Karolinska University Hospital-Solna, S-171 76 Stockholm, Sweden2 Bristol-Myers Squibb, Princeton, NJ
aAuthor for correspondence. Fax 46-8-5177-4245; e-mail Suzanne.Egyhazi{at}onkpat.ki.se.
To the Editor:
Gene expression profiling by microarray technology has demonstrated the possibilities of biological separation, prognostication, and prediction (1)(2). In this study we demonstrate that the addition of proteinase K to the extraction procedure improves the yield of RNA from primary breast tumors, making the majority of samples eligible for array analysis by Qiagen RNeasy Minikit. In addition, we investigated whether the addition of proteinase K has any influence on the gene expression profile.
The Qiagen RNeasy Mini protocol was used as described by the manufacturer (3)(4) for extraction of total RNA from 209 frozen primary breast tumors from women who had undergone surgery for breast cancer at Karolinska Hospital. The study was approved by an ethics committee at the hospital. Biopsies from the same tumors were extracted either with or without proteinase K treatment as follows. Each sample (maximum of 40 mg) was cut into smaller pieces and homogenized for 30–40 s in 400 µL of RLT lysis buffer containing mercaptoethanol with use of a polytron PT1200 homogenizer (Kinematica AG). For the proteinase K– sample, one volume (400 µL) of 700 g/L ethanol was added to the homogenate, with mixing during the addition, after which the sample was applied to a RNeasy mini spin column. For the proteinase K+ sample, 785 µL of doubly distilled water plus 13.5 µL of proteinase K (20 g/L; Qiagen GmbH) was added to the homogenate, which was then mixed and incubated for 10 min at 55 °C. The proteinase K+ sample was then centrifuged at 12 000g for 2 min, and 600 µL of absolute ethanol was added to the supernatant, with mixing during the addition. The sample was then applied to a RNeasy mini spin column. From this point, the same procedure was followed for both the proteinase K– and proteinase K+ samples. The column was washed once with 350 µL of RW1 buffer followed by on-column treatment with 80 µL of DNase solution (10 µL of DNase + 70 µL of RDD buffer from the Qiagen RNase-free DNase reagent set) for 15 min, according to the Qiagen recommendations. The column was then washed once with 350 µL of RW1 and twice with 500 µL of RPE buffer. Finally, the RNA was eluted with 50 µL of RNase-free water. We assessed the quality of the RNA by measuring the 28S:18S ribosomal RNA ratio with the Agilent 2100 Bioanalyzer (Agilent Technologies).
Proteinase K treatment increased the mean yield of RNA more than 10-fold, from 1.54 µg (range, 0–64.3 µg) to 20.3 µg (range, 0–125.6 µg). The proteinase K treatment increased the number of samples that could be used for microarray analyses from 10 of 209 to 152 of 209 samples (P <0.0001, Fisher exact two-tailed test) with 2 µg as the minimum amount of RNA per tumor required for the microarray studies.
To determine whether the addition of proteinase K has an effect on the gene expression profile, we performed microarray analyses according to the Affymetrix protocol, using Affymetrix HG-U133 (45 000 probe sets) chips because Affymetrix array results have shown high reproducibility (5). In two cases (biopsies 19 and 216), we performed gene expression analyses on two adjacent pieces from the same tumor, either including or excluding proteinase K in the RNA extraction procedure. The pairwise correlation coefficients for tumors 19 and 216 were 0.94 and 0.95, respectively, based on signal intensities of the probe sets, indicating that proteinase K treatment does not change the expression profile, whereas it increases the yield of RNA by making the tumor cells more accessible for extraction.
In summary, proteinase K treatment dramatically improves the yield of RNA without causing any significant changes in the expression profile when included in the Qiagen RNeasy protocol and markedly increases the amount of RNA purified from primary breast tumors by this method.
Acknowledgments
Our research groups are supported by grants from the Swedish Cancer Society, the Stockholm Cancer Society, King Gustav V’s Jubilee Fund, and Bristol-Myers Squibb.
References
The following articles in journals at HighWire Press have cited this article:
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  C. I. Dumur, M. Lyons-Weiler, C. Sciulli, C. T. Garrett, I. Schrijver, T. K. Holley, J. Rodriguez-Paris, J. R. Pollack, J. L. Zehnder, M. Price, et al. Interlaboratory Performance of a Microarray-Based Gene Expression Test to Determine Tissue of Origin in Poorly Differentiated and Undifferentiated Cancers J. Mol. Diagn., January 1, 2008; 10(1): 67 - 77. [Abstract] [Full Text] [PDF]
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 J Bergqvist, J. Ohd, J Smeds, S Klaar, J Isola, H Nordgren, G. Elmberger, H Hellborg, J Bjohle, A-L Borg, et al. Quantitative real-time PCR analysis and microarray-based RNA expression of HER2 in relation to outcome Ann. Onc., May 1, 2007; 18(5): 845 - 850. [Abstract] [Full Text] [PDF]
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