One of the difficulties encountered in studying rejection in patients is the availability of tissue. The goal of our study was to isolate RNA from archival allograft tissue, and to demonstrate that it is of suitable quality for further molecular experimentation.
Thirty-seven allograft archival samples were obtained from 18 patients (13 cardiac/5 renal transplant patients), based on a three year retrospective search of our University teaching hospitals tissue banks. RNA was extracted from the paraffin blocks and amplified by reverse-transcription-polymerase chain reaction (RT-PCR). Grade 3A and higher and non-rejection samples were tested. In addition, normal mouse liver tissue was isolated for comparison. Negative control samples were also included. Thirty-two cardiac biopsy samples were analyzed. Patient ages ranged from 15–67 years with a mean of 49.2 ± 15.1 SD. The average age of the blocks used was 2.0 years ± 1.29 SD (range from 13 days to 3.5 years in age). Renal allograft samples were also obtained for comparison, since renal biopsy tissue samples tend to be larger than endomyocardial samples.
Pathology slides were assigned histologic grades using the International Society for Heart and Lung Transplantation (ISHLT) 7 scale, and then compared to the original report after unblinding. All specimens had the same grade as the original pathology report. RT-PCR amplification of 18s RNA, 324 bp target sequence, revealed readily detectable bands. Our study showed a higher than expected yield from 31 samples studied, based on semi-quantitative RT-PCR analysis. Only one block, three years old, did not yield detectable RNA secondary to presumed degradation.
We conclude that RNA from archived allograft tissue can be used for further experiments. The use of this tissue offers distinct advantages when correlating gene expression with clinical outcome and therapeutic response. Formalin-fixed, paraffin-embedded tissue represents widely available clinical material for retrospective patient studies.
These tissues are an invaluable resource for the elucidation of rejection mechanisms, and exploration of differentially expressed genes as novel therapeutic targets or prognostic indicators.
A. Dosanjh, None.