Heparin interference with reverse transcriptase polymerase chain reaction of RNA extracted from lungs after ischemia-reperfusion.
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-a (TNF-a) 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.
Bai
X
X
The Toronto Lung Transplant Program and The Thoracic Surgery Research Laboratory, The Toronto General Hospital, Ontario, Canada.
Fischer
S
S
Keshavjee
S
S
Liu
M
M
D000818:Animals; D000925:Anticoagulants / Q000008:administration & dosage / Q000009:adverse effects*; D016207:Cytokines / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods; D006493:Heparin / Q000008:administration & dosage / Q000009:adverse effects*; D008168:Lung / Q000187:drug effects* / Q000473:pathology / Q000502:physiology; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury; D012680:Sensitivity and Specificity
D000925:Anticoagulants; D016207:Cytokines; D006493:Heparin