The microextraction of RNA from archival cardiac allografts embedded in paraffin.
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.
Bledsoe
Brian
B
Pediatric Oncology Education Program, St Jude Children's Research Hospital, Memphis, TN, USA.
Groshart
Kenneth
K
Zhang
Quaing
Q
Quasney
Michael
M
Dosanjh
Amrita
A
D000293:Adolescent; D000328:Adult; D000368:Aged; D000818:Animals; D020029:Base Pairing / Q000235:genetics; D005260:Female; D006084:Graft Rejection / Q000235:genetics / Q000473:pathology; D016027:Heart Transplantation / Q000473:pathology*; D006801:Humans; D016030:Kidney Transplantation / Q000473:pathology; D008099:Liver / Q000033:anatomy & histology / Q000378:metabolism; D008297:Male; D051379:Mice; D008832:Microchemistry; D008875:Middle Aged; D016612:Paraffin Embedding; D012313:RNA / Q000032:analysis*; D012337:RNA, Ribosomal, 18S / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013997:Time Factors; D014184:Transplantation, Homologous
D012337:RNA, Ribosomal, 18S; D012313:RNA