Optimization of an RNA isolation procedure from plasma samples.
In this study, we aimed to optimize the isolation procedure of circulating RNA from large volumes of plasma. Simultaneously, the stability and integrity of RNA from plasma samples were examined. To investigate the isolation of circulating RNA, reverse transcription-PCR analysis in combination with capillary electrophoresis was used. The presence of amplifiable RNA in plasma from healthy volunteers and from breast cancer patients was analyzed. We found that circulating RNA in plasma was highly fragmented and degraded. Plasma RNA was most efficiently isolated from large volumes of samples after introducing the step of plasma concentration by evaporation and by using TRIzol LS reagent. A single freeze/thaw process had no significant effect on RNA integrity and quantity of plasma RNA. The average amount of RNA in plasma from breast cancer patients was lower than in plasma from healthy volunteers. The concentrating of large volumes of plasma facilitates isolation of plasma RNA and yields amplifiable RNA for at least fragments that are up to 310 bp long.
Department of Molecular Diagnostics, Institute of Oncology Ljubljana, 1000 Ljubljana, Slovenia.
Petra
P
Cerkovnik
Andraz
A
Perhavec
Janez
J
Zgajnar
Srdjan
S
Novakovic
D000328:Adult; D001483:Base Sequence; D001943:Breast Neoplasms / Q000097:blood / Q000235:genetics; D045744:Cell Line, Tumor; D017931:DNA Primers / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods; D005260:Female; D005615:Freezing; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D011163:Hydroxymethylbilane Synthase / Q000235:genetics; D008297:Male; D008875:Middle Aged; D010949:Plasma / Q000737:chemistry; D012313:RNA / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification*; D020871:RNA Stability; D012334:RNA, Neoplasm / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D012334:RNA, Neoplasm; D012313:RNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D011163:Hydroxymethylbilane Synthase