Optimized lymphocyte protein extraction performed simultaneously with DNA and RNA isolation: application to the study of factors affecting DNA, RNA, and protein recovery from lymphocytes of the oldest individuals.
We describe an optimized procedure for protein extraction performed simultaneously with that of DNA and RNA from a single tissue sample that is, unlike the original protocol, suitable for quantitative studies. This optimized protocol is particularly well adapted to studies where gene regulation at DNA, RNA, and protein levels must be examined simultaneously, and when the amount of starting biological material is limited. We applied this procedure to the study of factors affecting both qualitatively and quantitatively the extraction of DNA, RNA, and proteins from lymphocytes of very old individuals, since we observed variability in the recovery of these molecular species with advanced age. Therefore, we investigated the combined effects of age and time delay between blood collection and lymphocyte isolation on the recovery of DNA, RNA, and proteins simultaneously extracted from Danish nonagenarians and centenarians versus younger adult samples. Our results suggest that neither RNA nor DNA nor protein contents of lymphocytes are altered with aging. However, the quantity of RNA and protein recovery is affected by a 24-h delay in blood processing. This effect is more pronounced in the oldest, particularly for RNA, and may affect data interpretation of age-dependent gene expression studies.
Riol
H
H
Lady Davis Institute, Jewish General Hospital, Montréal, Québec, Canada.
Jeune
B
B
Moskovic
A
A
Bathum
L
L
Wang
E
E
D016475:3T3 Cells; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001798:Blood Proteins / Q000302:isolation & purification* / Q000378:metabolism; D004247:DNA / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism*; D008297:Male; D051379:Mice; D008875:Middle Aged; D012313:RNA / Q000302:isolation & purification*; D013048:Specimen Handling
D001798:Blood Proteins; D012313:RNA; D004247:DNA