Quantitative mRNA Expression Analysis from Formalin-Fixed, Paraffin-Embedded Tissues Using 5′ Nuclease Quantitative Reverse Transcription-Polymerase Chain.

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Presentation transcript:

Quantitative mRNA Expression Analysis from Formalin-Fixed, Paraffin-Embedded Tissues Using 5′ Nuclease Quantitative Reverse Transcription-Polymerase Chain Reaction Tony E. Godfrey, Sun-Hun Kim, Marielena Chavira, David W. Ruff, Robert S. Warren, Joe W. Gray, Ronald H. Jensen The Journal of Molecular Diagnostics Volume 2, Issue 2, Pages 84-91 (May 2000) DOI: 10.1016/S1525-1578(10)60621-6 Copyright © 2000 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Denaturing agarose gel of RNA isolated from 3 archival prostate tissue blocks collected in 1985, 1990, and 1993, respectively. RNA isolated using the modified 3-day protocol is longer (A) and the yield is higher (B) than with the original protocol. The Journal of Molecular Diagnostics 2000 2, 84-91DOI: (10.1016/S1525-1578(10)60621-6) Copyright © 2000 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Amplification of different sized fragments of the β-actin mRNA from fixed tissue RNA isolates. Greatly increased sensitivity of detection is obtained with fragments less than 131 basepairs. The forward primer and TaqMan probe were kept constant and used with different reverse primers to change amplicon length (inset picture). No difference is seen between the immediate fix RNA and the 12 hour pre-fixation RNA samples (see Materials and Methods for description of tissue processing). Fresh tissue RNA was used as a high quality RNA control. Standard deviations on all points were smaller than the size of the symbols and are omitted. The Journal of Molecular Diagnostics 2000 2, 84-91DOI: (10.1016/S1525-1578(10)60621-6) Copyright © 2000 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Relative abundance of 99- and 291-bp fragments of the β-actin mRNA in fixed tissues. All points are measured relative to β-Gus and are normalized to the fresh tissue RNA. The Journal of Molecular Diagnostics 2000 2, 84-91DOI: (10.1016/S1525-1578(10)60621-6) Copyright © 2000 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Effect of pre-fixation time on quantitation of six different mRNA species. All points are measured relative to β-Gus and are normalized to the fresh tissue RNA. The Journal of Molecular Diagnostics 2000 2, 84-91DOI: (10.1016/S1525-1578(10)60621-6) Copyright © 2000 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 Effect of fixation and pre-fixation time on quantitation of different pieces of the c-myc mRNA. Primers and probe used are shown in Table 1. Fragments are affected differently by fixation but none of the fragments show a change in abundance over the 12-hour time series. All points are measured relative to β-Gus and are normalized to the fresh tissue RNA. The Journal of Molecular Diagnostics 2000 2, 84-91DOI: (10.1016/S1525-1578(10)60621-6) Copyright © 2000 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions