Concentrations of circulating RNA from healthy donors and cancer patients estimated by different methods.
Circulating RNA (cirRNA) was isolated from plasma and cell surface-bound fractions of blood of healthy women and breast cancer patients. RNA samples were DNase treated and quantified by a SYBR Green II assay. Concentrations of RNA sequences of GAPDH, Ki-67 mRNA, and 18S rRNA were measured by real-time quantitative PCR (RT-qPCR) after reverse transcription with random hexamer primers. The obtained data spread over three orders of magnitude for GAPDH and Ki-67 mRNA signals and two orders of magnitude for the copy number of 18S rRNA in blood fractions in both groups. In blood of healthy donors, no correlation was found between the copy number of GAPDH, Ki-67 mRNA, and 18S rRNA and RNA concentrations measured by the SYBR Green II assay. Within the group of breast cancer patients, the concentration GAPDH and Ki-67 mRNA correlated with the concentration of total RNA only in the cell surface-bound fraction; whereas the concentration of 18S rRNA correlated with total RNA in both, the cell surface-bound fraction and blood. The copy number of Ki-67 mRNA correlated with copy numbers of GAPDH mRNA in all fractions of cirRNA of healthy donors and breast cancer patients. A correlation between copy numbers of Ki-67 mRNA and 18S rRNA was found only in cell surface-bound fraction of breast cancer patients. The data described here demonstrate the necessity of searching for more suitable RNA markers in order to estimate total cirRNA concentrations by RT-qPCR, although mRNA of GAPDH could be used for normalization of the level of cancer-specific mRNA among patients.
Rykova
Elena Y
EY
Institute of Chemical Biology and Fundamental Medicine SB of RAS, Lavrentiev Ave. 8, Novosibirsk, Russia. rykova@niboch.nsc.ru
Wunsche
Winfried
W
Brizgunova
Olga E
OE
Skvortsova
Tatyana E
TE
Tamkovich
Svetlana N
SN
Senin
Ilija S
IS
Laktionov
Pavel P
PP
Sczakiel
Georg
G
Vlassov
Valentin V
VV
D014408:Biomarkers, Tumor / Q000097:blood / Q000235:genetics; D001943:Breast Neoplasms / Q000097:blood / Q000235:genetics; D005260:Female; D005819:Genetic Markers; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000097:blood / Q000235:genetics; D006801:Humans; D019394:Ki-67 Antigen / Q000097:blood / Q000235:genetics; D012313:RNA / Q000097:blood*; D012337:RNA, Ribosomal, 18S / Q000097:blood; D013223:Statistics as Topic
D014408:Biomarkers, Tumor; D005819:Genetic Markers; D019394:Ki-67 Antigen; D012337:RNA, Ribosomal, 18S; D012313:RNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases