Effects of Oligo dT-T7 RNA Primer in RNA Amplification from Paraffin-Embedded Tissue for Microarray Experiments

ABSTRACT

Introduction: Formalin-Fixed Paraffin-Embedded Tissue samples (FFPET) represent a valuable source for studies of geneexpression comparisons, since a great number of these samples is available in archive and presents a long time of clinicalfollow-up. However, the quality of total RNA of these samples is known to be inferior to frozen samples, being many timesinadequate for studies of gene expression using conventional methodologies. Objective: This study aims to establish a protocolfor amplification of messenger RNA (mRNA) derived from FFPET samples for using in microarray experiments. Material andMethods: 4 tumoral samples of invasive ductal breast carcinoma FFPET-buffered 10% were used. Total RNA was extracted andthe mRNA was linearly amplified in two rounds based on T7 RNA polymerase methodology using different concentrations ofoligo dT-T7 Primer for first strand cDNA (1st-cDNA) synthesis. Amplified antisense RNA (aRNA) was labeled with cianine-Cy3through reverse transcription in the presence of random primers and co-hybridized with reference RNA (HB4a) labeled withcianine-Cy5 in a customized platform containing 4,608 cDNAs corresponding to human genes. Results: The amplified RNAquality was influenced by the relative amount of oligo dT-T7, showing better results for ratio of 10.1 (total RNA oligo dT-T7).Hybridizations showed value of intensity signals for the most of cDNAs immobilized in the platform. Conclusion: This studyshowed that the control of the relative amounts of RNA derived from FFPET material and oligo dT-T7 is extremely important toobtain high-quality amplified RNA, allowing its use in microarray experiments.