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Updated: Aug 22, 2026
Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens
Published on: May 5, 2018
The microextraction of RNA from archival cardiac allografts embedded in paraffin
Brian Bledsoe1, Kenneth Groshart, Quaing Zhang
1Pediatric Oncology Education Program, St Jude Children's Research Hospital, Memphis, TN, USA.
Abstract:
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-two paraffin embedded cardiac and five renal allograft archival samples were obtained after IRB approval, from a total of 18 transplant patients (13 cardiac/five renal transplant patients) from a search of the University of Tennessee's teaching hospitals. 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. RT-PCR amplification of 18s RNA, 324 bp target sequences, revealed readily detectable bands. Only one block that was 3 years old did not yield detectable RNA secondary to presumed degradation. The negative control showed no bands at 324 bp. We conclude that RNA from archived allograft tissue can be used for further experiments. The use of this tissue offers some distinct advantages when studying correlation of gene expression with clinical outcome and therapeutic response.
Insights
Archival allograft tissue yields high-quality RNA suitable for molecular studies. This discovery enables research correlating gene expression with transplant outcomes and therapeutic responses, overcoming tissue availability challenges.
Area of Science:
- Transplantation immunology
- Molecular biology
- Genomics
Background:
- Studying organ transplant rejection is hindered by limited tissue availability.
- Archival tissue offers a potential resource for molecular analysis.
- Assessing RNA quality from archival samples is crucial for downstream applications.
Purpose of the Study:
- To isolate RNA from archival allograft tissue.
- To confirm the suitability of extracted RNA for molecular experimentation.
- To explore the utility of archival tissue in transplant research.
Main Methods:
- RNA extraction from 32 paraffin-embedded cardiac and 5 renal allograft samples.
- Reverse transcription-polymerase chain reaction (RT-PCR) amplification of 18s RNA.
- Testing of Grade 3A and higher, non-rejection, and control samples.
Main Results:
- Readily detectable bands were observed for 18s RNA (324 bp) via RT-PCR.
- RNA was successfully extracted from most archival samples, including those up to 3 years old.
- Negative controls showed no detectable bands, confirming assay specificity.
Conclusions:
- RNA isolated from archival allograft tissue is of sufficient quality for molecular experiments.
- Archival tissue provides a valuable, accessible resource for transplant research.
- This method facilitates the correlation of gene expression with clinical outcomes and treatment responses.
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