Open AccessHeparin interference with reverse transcriptase polymerase chain reaction of RNA extracted from lungs after ischemia‐reperfusion
Author(s) -
Bai Xiaohui,
Fischer Stefan,
Keshavjee Shaf,
Liu Mingyao
Publication year - 2000
Publication title -
transplant international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.998
H-Index - 82
eISSN - 1432-2277
pISSN - 0934-0874
DOI - 10.1111/j.1432-2277.2000.tb01055.x
Subject(s) - medicine , reverse transcriptase , heparin , polymerase chain reaction , reverse transcription polymerase chain reaction , ischemia , messenger rna , gene , biochemistry , biology
The reverse transcriptase polymerase chain reaction (RT‐PCR) is a rapid and sensitive method for detecting gene expression. However, when we used this technique to study gene expression of cytokines in ischemic and ex‐vivo‐reperfused rat lungs as a model for transplantation, significant inhibition of RT‐PCR reaction was observed. To optimize RT‐PCR conditions, RNA was extracted from rat lungs after flushing, preservation, and reperfusion. RNA was further purified and PCR conditions were modified with various strategies. We found that heparinase I pretreatment completely overcame the inhibitory effects of RT‐PCR using RNA extracted from lung tissues after ischemia‐reperfusion. With this treatment, a dramatic increase in tumor necrosis factor‐α (TNF‐α) mRNA was revealed from lung tissues after ischemia‐reperfusion. This result suggests that residual heparin in lung tissue interferes with RT‐PCR. Because heparinization is routinely used during clinical and experimental organ transplantation, we recommend the treatment of RNA samples with heparinase prior to RT‐PCR.