Journal: Nucleic Acids Research
Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)
doi: 10.1093/nar/gkm510
Figure Lengend Snippet: Experimental procedure utilized for the analysis of 10-year-old matched frozen and formalin-fixed paraffin embedded breast cancer samples. ( a ) Five micrograms of RNA extracted from the 10-year-old frozen portion of the sample, is reverse-transcribed and the cDNA is double stranded (dsDNA), in four individual reactions. The dsDNA of three reactions undergoes IVT-amplification (MessageAmpII, Ambion), which gives rise to complementary RNA (cRNA) for cDNA microarray analyses. The dsDNA of one reaction is used for PCR experiments. ( b ) Five micrograms of RNA extracted from the 10-year-old FFPE portion of the sample underwent the exact same process. ( c ) Single-stranded DNA (ssDNA) obtained by RT of 5 μg of FFPE-RNA is purified and hybridized to the sense-RNA template library. The restored ssDNA is double stranded and purified. Three of the CT-RT reactions undergo IVT-amplification, while the dsDNA of one reaction is used for PCR experiments.
Article Snippet: Arrays used for the studies were designed and printed at the cDNA Microarray Facility, Albert Einstein College of Medicine (AECOM), Bronx, NY.
Techniques: Formalin-fixed Paraffin-Embedded, Reverse Transcription, Amplification, Microarray, Purification