Michelle Mandrekar

Abstract 4846: Automated RNA and DNA purification from FFPE samples

Formalin-fixed, paraffin-embedded (FFPE) samples are commonly used for archiving pathology samples for use by many researchers including clinical research labs. These samples provide a valuable tool for retrospective studies of diseases such as cancer. Traditional methods for the purification of DNA or RNA from FFPE tissue samples are often labor intensive, include the use of hazardous organic reagents, and involve difficult pre-processing steps. Here, we describe development of automated methods for the purification of RNA or DNA from FFPE tissue sections using the Maxwell® RSC Instrument, which can process between 1 and 16 samples. DNA was purified from FFPE mouse brain, liver, lung, skin and spleen as well as FFPE human colon. RNA was purified from FFPE mouse brain, liver, lung, skin and spleen as well as FFPE human breast tissue. Eluates were tested for inhibition in qPCR or RT-qPCR. A comparison with another automated method was performed with FFPE mouse spleen and skin and a human tissue. The Maxwell RSC methods produced high quality amplifiable DNA or RNA from a variety of sample types with little or no inhibition. In addition, these methods simplified pre-processing, minimized hands-on time, and did not use hazardous organics. Citation Format: Michelle Mandrekar, Douglas Horejsh, Samantha Lewis, Chris Moreland, Marjeta Urh. Automated RNA and DNA purification from FFPE samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4846. doi:10.1158/1538-7445.AM2015-4846

Reversed enzyme activity DNA interrogation test: a new method for mutation detection

The Reversed Enzyme Activity DNA Interrogation Test (READITTM) System provides a new, rapid method for genotype determination. The system can be used for the analysis of single nucleotide polymorphisms as well as insertions, deletions and chromosomal rearrangements. The READITTM System is based upon driving various enzymatic reactions in the reverse direction from the direction they are normally considered. The method is based on a pyrophosphorylation reaction that is the reverse of DNA polymerization. Interrogation probes annealed to DNA fragments with which they have a 3- mismatch are not a substrate for the reaction and do not generate significant signals compared with probes that match the interrogation sequence perfectly. By interrogation of specific DNA sequences with carefully designed probes, mutant genes can be identified with speed and precision.