Volume 1, Issue 2, Pages 72-80 (November 2009)

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ABSTRACT

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A Novel Technique for Detecting the Therapeutic Target, KRAS Mutant, From Peripheral Blood Using the Automatic Chipball Device With Weighted Enzymatic Chip Array

Received 22 October 2009; received in revised form 16 November 2009; accepted 18 November 2009.

Reverse transcriptase and real-time polymerase chain reactions are widely used for the detection of gene overexpression. However, various disadvantages and limitations arise when the detection of multiple genetic targets is required. In previous studies, our laboratory successfully established a membrane array operation platform with a diagnostic biochip for the screening of gene overexpression by circulating tumor cells in cancer patients. To effectively shorten the reaction time, we improved the conventional RNA extraction method. The concept of weightedness was included in the reading procedure of the chip array and a weighted enzymatic chip array (WEnCA) platform was established. We used fluid engineering to develop a fully automatic gene chip analyzer named Chipball, which runs automatically on the WEnCA platform. The combination of the two systems is named the WEnCA-Chipball system. To understand the actual differences between the operations of WEnCA-Chipball and WEnCA-manual, we used the WEnCA-manual to analyze KRAS-associated gene overexpression in 200 samples from cancer patients to establish a cutoff value for activating the KRAS Detection Chip. Specifically, the activated KRAS expression in blood samples of 209 lung cancer patients was analyzed by both WEnCA-manual and WEnCA-Chipball and compared. The clinical applicability of WEnCAChipball was defined, including the sensitivity, specificity, and accuracy. The results showed that among 209 samples, 71 patients were positive for activated KRAS expression by WEnCA-Chipball with a sensitivity of 89%, specificity of 94%, and accuracy of 92%. In addition, the average total score of WEnCA-Chipball was 4.7 lower than that of the WEnCA-manual. The WEnCA-Chipball required an operation time of only 7.5 hours, approximately one-ninth of the WEnCA-manual operation time and one-fifth of the cost of WEnCA-manual. No significant difference was found between the detection limitations of the two systems. Great strides have been made in this development. The WEnCA-Chipball operation system has potential for clinical applications.

Key Words: Activating KRAS Detection Chip , WEnCA-manual , WEnCA-Chipball

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Article Outline

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References

1. 1 Yu M , Wan Y , Zou Q . Prognostic significance of BP1 mRNA expression level in patients with non-small cell lung cancer . Clin Biochem . 2008;41:824–830 . CrossRef

2. 2 Wu CH , Lin SR , Yu FJ , et al. Development of a high-throughput membrane-array method for molecular diagnosis of circulating tumor cells in patients with gastric cancers . Int J Cancer . 2006;119:373–379 . MEDLINE | CrossRef

3. 3 Poulsen TT , Pedersen N , Perin MS , et al. Specific sensitivity of small cell lung cancer cell lines to the snake venom toxin taipoxin . Lung Cancer . 2005;50:329–337 . Abstract | Full Text | Full-Text PDF (325 KB) | CrossRef

4. 4 Wu CH , Lin SR , Hsieh JS , et al. Molecular detection of disseminated tumor cells in the peripheral blood of patients with gastric cancer: evaluation of their prognostic significance . Dis Markers . 2006;22:103–109 . MEDLINE

5. 5 Saito D , Coutinho LL , Borges Saito CP , et al. Real-time polymerase chain reaction quantification of Porphyromonas gingivalis and Tannerella forsythia in primary endodontic infections . J Endod . 2009;35:1518–1524 . Abstract | Full Text | Full-Text PDF (139 KB) | CrossRef

6. 6 Yang M , Cheng A , Wang M , et al. Development and application of a one-step real-time TaqMan RT-PCR assay for detection of Duck hepatitis virus type 1 . J Virol Methods . 2008;153:55–60 . CrossRef

7. 7 Karakoc AE , Berkem R , Beyaz E . Detection of hepatitis B virus deoxyribonucleic acid using real-time polymerase chain reaction method in pooled-plasma samples of Turkish blood donors . Clin Lab . 2009;55:229–234 .

8. 8 Böhl M , Schwenzer B . A potent inhibitor of prothrombin gene expression as a result of standardized target site selection and design of antisense oligonucleotides . Oligonucleotides . 2005;15:172–182 . MEDLINE | CrossRef

9. 9 Ball TB , Plummer FA , HayGlass KT . Improved mRNA quantitation in LightCycler RT-PCR . Int Arch Allergy Immunol . 2003;130:82–86 . MEDLINE | CrossRef

10. 10 Walder RY , Hayes JR , Walder JA . Use of PCR primers containing a 3'-terminal ribose residue to prevent cross-contamination of amplified sequences . Nucleic Acids Res . 1993;21:4339–4343 . MEDLINE

11. 11 Popovtzer R , Neufeld T , Popovtzer A , et al. Electrochemical lab on a chip for high-throughput analysis of anticancer drugs efficiency . Nanomedicine . 2008;4:121–126 . Abstract | Full Text | Full-Text PDF (534 KB) | CrossRef

12. 12 Chen CC , Hou MF , Wang JY , et al. Simultaneous detection of multiple mRNA markers CK19, CEA, c-Met, Her2/neu and hMAM with membrane array, an innovative technique with a great potential for breast cancer diagnosis . Cancer Lett . 2006;240:279–288 . Abstract | Full Text | Full-Text PDF (302 KB) | CrossRef

13. 13 Chen CC , Chang TW , Chen FM , et al. Combination of multiple mRNA markers (PTTG1, Survivin, UbcH10 and TK1) in diagnosis of Taiwanese patients with breast cancer by membrane array . Oncology . 2006;70:438–446 .

14. 14 Tsao DA, Yang MJ, Chang HJ, et al. A fast and convenient new technique to detect the therapeutic target, K-ras mutant, from peripheral blood in non-small cell lung cancer patients. Lung Cancer 2009 (In press).

15. 15 Matsugi J , Murao K . Study on construction of a cDNA library corresponding to an amino acid-specific tRNA and influence of the modified nucleotide upon nucleotide misincor-porations in reverse transcription . Biochim Biophys Acta . 2001;1521:81–88 . MEDLINE

16. 16 Zhu Q , von Dippe P , Xing W , et al. Membrane topology and cell surface targeting of microsomal epoxide hydrolase: evidence for multiple topological orientations . J Biol Chem . 1999;274:27898–27904 . MEDLINE | CrossRef

17. 17 Harder N , Mora-Bermúdez F , Godinez WJ , et al. Automatic analysis of dividing cells in live cell movies to detect mi-totic delays and correlate phenotypes in time . Genome Res . 2009;19:2113–2124 . CrossRef

18. 18 Sajda P . Machine learning for detection and diagnosis of disease . Annu Rev Biomed Eng . 2006;8:537–565 . MEDLINE | CrossRef

19. 19 Sobrero A , Aschele C , Guglielmi A , et al. Resistance to 5-fluorouracil and 5-fluoro-2'-deoxyuridine mechanisms and clinical implications . J Chemother . 1990;2(Suppl 1):S12–S16 .

20. 20 Rossi A , Maione P , Palazzolo G , et al. New targeted therapies and small-cell lung cancer . Clin Lung Cancer . 2008;9:271–279 . CrossRef

21. 21 Wu JT , Kral JG . The NF-κB/IκB signaling system: a molecular target in breast cancer therapy . J Surg Res . 2005;123:158–169 . Abstract | Full Text | Full-Text PDF (335 KB) | CrossRef

22. 22 Modjtahedi H , Essapen S . Epidermal growth factor receptor inhibitors in cancer treatment: advances, challenges and opportunities . Anticancer Drugs . 2009;20:851–855 . CrossRef

23. 23 Funfak A , Brösing A , Brand M , et al. Micro fluid segment technique for screening and development studies on Danio rerio embryos . Lab Chip . 2007;7:1132–1138 .

24. 24 Duenwald S , Zhou M , Wang Y , et al. Development of a microarray platform for FFPET profiling: application to the classification of human tumors . J Transl Med . 2009;7:65 .

25. 25 Ohno K , Tachikawa K , Manz A . Microfluidics: applications for analytical purposes in chemistry and biochemistry . Electrophoresis . 2008;29:4443–4453 . CrossRef

a School of Medical and Health Science, Fooyin University, Kaohsiung, Taiwan

b Department of Medical Research, Fooyin University Hospital, Pingtung, Taiwan

c Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

d Department of Surgery, Fooyin University Hospital, Pingtung, Taiwan

e School of Environmental and Life Sciences, Fooyin University, Kaohsiung, Taiwan

f Technology Development Center of Molecular Diagnosis and Fermentation Engineering, Fooyin University, Kaohsiung, Taiwan

g Gene Target Technology Co. Ltd., Kaohsiung, Taiwan

h Faculty of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan

Corresponding authors. Tian-Lu Cheng: Faculty of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan. Shiu-Ru Lin: School of Medical and Health Sciences, Fooyin University, 151 Chin-Hsueh Road, Ta-Liao Hsiang, Kaohsiung 831, Taiwan

† Suz-Kai Hsiung and Hui-Jen Chang contributed equally to this work.

PII: S1877-8607(10)60003-3

doi:10.1016/S1877-8607(10)60003-3

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