A simple, cost-effective and flexible method for processing of snap-frozen tissue to prepare large amounts of intact RNA using laser microdissection.
Understanding the molecular basis of disease requires gene expression profiling of normal and pathological tissue. Although the advent of laser microdissection (LMD) has greatly facilitated the procurement of specific cell populations, often only small amounts of low quality RNA is recovered. This precludes the use of global approaches of gene expression profiling which require sizable amounts of high quality RNA. Here we report a method for processing of snap-frozen tissue to prepare large amounts of intact RNA using LMD. Portions of small intestine from piglets (n = 6) were snap-frozen in Optimum Cutting Temperature compound (experimental) and in RNAlater (control). A randomly selected sample was laser microdissected using the developed protocol in multiple sessions totalling 4 h each day on four consecutive days. RNAs were extracted from these samples and its control and their quality (RIN) determined. RINs of the experimental samples were independent of time (p = 0.12) and day (p = 0.56) of the microdissection thereby suggesting that their RNA quality remained unaltered. These samples exhibited high quality (RIN ≥ 8) with good recovery (81.2%) and excellent yield (1539 ng/1.2 × 10(7) μm(2)). Their overall RIN, 8.029 ± 0.116, was not significantly different from 8.2 (p = 0.123), the value obtained from the control, non-laser microdissected, sample. This indicated that the RNA quality from the laser microdissected and non-microdissected samples was comparable. The method allowed LMD for up to 4 h each day for a total of four days. The microdissected samples can be pooled thereby increasing amount of RNA at least by ten-fold. The procedure did not require any expensive limited-shelf life RNase inhibitors, RNA protectors, staining kits or toxic chemicals. Furthermore, it was flexible and enabled the processing without affecting routine laboratory workflow. The method developed was simple, inexpensive and provided substantial amounts of high quality RNA suitable for gene expression profiling and other cellular and molecular analyses for biology and molecular medicine.
Department of Surgery, Basil Hetzel Institute for Translational Health Research, The Queen Elizabeth Hospital and Discipline of Medicine, University of Adelaide, 28 Woodville Road, Woodville South, South Australia 5011, Australia. Phulwindergrover@yahoo.com
Phulwinder K
PK
Grover
Adrian G
AG
Cummins
Timothy J
TJ
Price
Ian C
IC
Roberts-Thomson
Jennifer E
JE
Hardingham
D000818:Animals; D003362:Cost-Benefit Analysis; D015925:Cryopreservation / Q000191:economics / Q000379:methods*; D005629:Frozen Sections / Q000191:economics / Q000379:methods*; D006801:Humans; D007421:Intestine, Small / Q000378:metabolism; D007834:Lasers; D042282:Microdissection / Q000295:instrumentation / Q000379:methods*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020871:RNA Stability; D015203:Reproducibility of Results; D034421:Sus scrofa; D013997:Time Factors
D012313:RNA