1. Improved PCR quantification of tumour-derived cDNA: ready-to-go template conditioning Introduction Quantification analyses for mRNA markers are an essential prerequisite for diagnosis and monitoring of tumour diseases or for new target-directed treatment strategies. Real-time PCR is a favourable option for the analysis of tumour-associated and tumour-specific transcripts [1]. As an enhancement to our previous studies describing the techniques of calibration and storage-stable coating of DNA standards [2] and their first application in molecular tumour diagnostics for prostate-associated transcripts and tumour-associated mRNA variants [3,4], we describe methodological aspects for preparation of robust ready-to-go cDNA samples generated from tumour cells and tumour tissues herein. We summarise our results for coating of cDNA samples of different sources in comparison to the identical cDNA samples diluted and discuss the benefits and possibilities for the management of multi-marker QPCR studies. Axel Meye, Anne-Kathrin Rost*, Susanne Fuessel, Uta Schmidt, Thomas Koehler*, Manfred P. Wirth Department of Urology, Technical University of Dresden & *Roboscreen GmbH, Leipzig, Germany Department of Urology, Technical University of Dresden & *Roboscreen GmbH, Leipzig, Germany Results & Discussion Sensitivity & reproducibility of external & coated DNA standards in glass capillaries limits for quantification of 10 molecules (for uPAR and trp-p8) & below (for PSA) could be determined for the coated DNA standards coating of standard DNA & template cDNA provides multiple advantages over the manual application of template DNA in solution: i) application of storage-stable DNA standards allows the realisation of large QPCR experiment series as validation parameter for calibration curves [2], ii) high reproducibility & thereby a good standardisation, iii) once prepared from a template source they can be used for QPCR studies covering a broad log scale spectrum (1x10E1 to 1x10E8 molecules) (Fig. 1) therefore, preparations of coated standards can be applied to study relatively high expression levels in matched tumour tissue pairs & cell lines as well as for the detection of minimal residual amounts of transcripts in lymph nodes or various body fluids reaction tubes coated with standards of up to 12 markers together were tested successfully without reduction of signal availability & amplification efficiency Ready-to-use cDNA templates: standardisation & improvement for screening studies different template preparations in optimised QPCR assays (Table I) for six different marker genes in different template dilution ranges (1:2 to 1:10) were compared; quantification results for different tumour-associated markers (PSA, trp-p8, uPAR) [3-6] and reference genes (GAPDH, PBGD, HPRT) [7-9] were comparable or even better (within-run and between-run variations of standards and samples) than those for manually diluted identical cDNA samples (Fig. 2); however, for the PSA and trp-p8 genes we detected a loss of PCR signals from day 56 onwards (Fig. 2A&B) but this lower availability of these assays was nearly equal for all three different types of cDNA storage results of trp-p8 QPCR for one cDNA sample coated into glass capillaries (n=6) or freshly diluted in water (n=3) derived from LNCaP were comparable in a cDNA dilution of 1:5 (2.561.94 x10E3 and 2.420.45 x10E3 template molecules) and of 1:10 (1.150.94 x10E3 & 1.221.06 x10E3 template molecules) results for PSA with cDNA samples derived from a PCa tissue showed that coating allows an increased template availability in comparison to freshly in water diluted cDNA samples for both the cDNA dilution of 1:5 (2.970.14 x10E4 vs. 2.060.09 x10E4 template molecules) & of 1:10 (1.320.07 x10E4 vs. 0.930.14 x10E4 template molecules) median PBGD expression data obtained from one coated cDNA (n=6) derived from RCC tissue specimen indicate higher template accessibility of 18% and 37% compared to the same cDNA sample diluted in water & stored at 4°C (n=3) & at -20°C (n=3) for 1 week in further series of experiments, the long-term stability of tissue-derived, coated cDNA samples was determined for different dilutions: storage of standards coated to glass capillaries and the diluted cDNA samples over a period of up to four months (in parallel at 4°C and –20°C) indicates the stability of coated cDNA template for QPCR (Fig. 2); interestingly, storage of templates as coated to glass capillaries and diluted cDNA stored at 4°C resulted in higher PCR signals than the storage as immediately frozen DNA solution over this period of time. in addition, the coating technique for cDNA samples of different origin is also applicable for QPCR systems based on plastic reaction tubes: for example, the transcript detection of the M-Bcr (median CT 28.33) and the GAPDH gene (median CT 22.35) was reproducible for a K562 cDNA samples (Table II). Conclusions conditioning & accessibility of coated cDNA for sensitive QPCR over several months generation of standardised positive controls is feasible one main advantage is the relatively high exploitation of template resources: limited human cell and tissue material as template source for QPCR studies can be isolated under standardised conditions and (immediately) confectioned without unnecessary loss of template due to multiple dilution processes ready-to-go confectioning of cDNA samples saves time in preparation of the PCR mix and reduces the risk of cross-contamination. this coating technique is particularly useful for multi-center studies (the ready-to-go templates can serve as internal & inter-center quality controls for each parameter) [12] template conditioning may serve as an additional validation platform in the guidance of new therapeutic modalities & the ability to detect & monitor MRD Material & Methods Tumour cells & tissues, RNA isolation, cDNA synthesis & coating 3 tissue specimens (from 1 PCa & 1 RCC tissue pair; samples of malignant tissue and corresponding non-malignant tissue) were collected during tumour surgery, snap frozen in liquid N2 slices (10-15µm) by a cryomicrotome, transfer in lysis buffer (Spin Tissue RNA Mini Kit, Invitek, Berlin, Germany) additional RNA samples from LNCaP & K562 cells cDNA synthesis using 1µg of total RNA in a 20µl standard reaction (200U Superscript II, Invitrogen) and random hexamers products of 6 RT reactions of the same sample were pooled & used for coating reaction vessels (glass capillaries & plastic tubes) using a slightly modified protocol as described [2] synthesised cDNAs were purified by phenol:chloroform:isoamyl alcohol (25:24:1) extraction and ethanol precipitation purified cDNA was resuspended in a patented carrier DNA- based stabilization solution (10ng/µl) aliquots of diluted cDNA (5µl) were freeze-dried in the capillaries or plastic tubes (Christ Gamma 1-20 lyophilisation apparatus, Christ GmbH, Osterrode, Germany) lyophilisation-mediated matrix formation between carrier DNA and cDNA allows improved target stability, complete desorption from the vial throughout subsequent PCR and complete availability of target to the amplification process QPCR analyses LightCycler technology (Roche, Mannheim, Germany) as described for the markers trp-p8 and uPAR [3,4, Table I] LC-QPCR 2µl of a defined dilution (1:5 or 1:10) of the respective cDNAs diluted in water & coated in glass capillaries capillaries storage at -20°C, samples of the identical cDNA dilutions were stored in plastic tubes at 4°C & at -20°C 2µl were pipeted into the glass capillaries immediately before the QPCR run and for defined times of investigation. mRNA copy number of a marker was calculated in relation to the amplification product amounts of external DNA standards absolute molecule numbers used for further calculations QPCR studies on coated plastic tubes using the RoboGene M- Bcr and GAPDH cDNA Quantification Modules (Roboscreen) [12]) long-term stability of coating to plastic tubes was tested by comparing the CT values for both genes over 270 days References [1]Bernard PS et al 2002. Clin Chem 48: 1178-1185. [2]Koehler T et al 1997. Biotechniques 23:722-726. [3]Fuessel S et al 2003. Int J Oncol 23: 21-29. [4]Luther T et al 2003. Thromb Haemastol 89:705-717. [5]Gao CL et al Clin Cancer Res 9: 2545-2450. [6]Tsavaler L et al 2001. Cancer Res. 61: 3760-3769. [7]Rey JM et al 2003. J Mol Endocrinol 24: 433-440. [8]Vandesompele J et al 2004. Genome Biol 3: RESEARCH0034. [9]Kuhne BS, Oschmann P 2002. Biotechniques 33: 1080-1082. [10]Ovstebo R et al Clin Chem 49: 425- 432. [11]O'Hara SM et al 2004. Clin Chem 50: 826-835. [12]Lange T. et al 2004. Haematologica 89: 49-57. #717 Figure 1Standard curves for PSA quantification using coated glass capillaries, where molecule numbers detected versus theoretical molecule numbers generated from eight independent PCR runs are shown. Figure 2Testing the stability of positive controls coated into glass capillaries with cDNA (at a dilution of 1:10) obtained from the prostate cancer cell line LNCaP. Indicated are the absolute molecule numbers. Table IPCR fragments, primers, and HP for QPCR Table IILong-term stability testing of cDNA obtained from the leukemic cell line K562 coated to plastic tubes. Median threshold cycle numbers and standard deviations (n=5) for the 2 target genes (M- Bcr, GAPDH) & the ratios of CT values are indicated Acknowledgments We are very thankful to Mrs. Andrea Lohse and Mrs. Katja Robel from the Department of Urology for excellent technical assistance.