ISSN:
1432-2277
Keywords:
Key words Reverse transcriptase polymerase chain reaction
;
Transplantation
;
Heparin
;
Ischemia-reperfusion
;
Gene expression
;
Cytokines
Source:
Springer Online Journal Archives 1860-2000
Topics:
Medicine
Notes:
Abstract 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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s001470050306
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