A simple procedure for routine RNA extraction and miRNA array analyses from a single thyroid in vivo fine needle aspirate.
BACKGROUND
microRNA (miRNA) expression profiling and classification of tissue obtained from fine-needle aspirates (FNA) could be a major improvement of the preoperative diagnosis of thyroid nodules.


OBJECTIVE
Before this can be clinically implemented, a robust and non-toxic method for obtaining sufficient quantity and quality of RNA from single in vivo FNA has to be established. RNAlater is a non-toxic RNA-stabilizing agent. However, due to the high density of RNAlater, pelleting of the tissue samples is difficult, and results in low recovery of RNA that is insufficient for subsequent miRNA array expression analysis. We therefore developed a simple centrifugation method for capturing tissue stored in RNAlater on a 0.45-μm filter.


METHODS
FNA from 24 patients with a solitary cold thyroid nodule was stored in Trizol, liquid nitrogen, or RNAlater. The tissue stored in RNAlater was either pelleted by centrifugation or captured on the 0.45-μm filters. RNA was extracted using the Trizol method. To validate results, FNA from additional 30 patients were analyzed based on the modified RNAlater protocol.


RESULTS
Capturing FNA tissue samples on the filters increased the RNA yield 10 fold and maintained RNA purity, permitting miRNA array expression profiling and allowing comparable levels of known miRNA-clusters regardless of preservation technique. Results were confirmed in an additional 30 patients.


CONCLUSIONS
The modified RNAlater protocol is well suited for isolating RNA from single thyroid in vivo FNA in a clinical setting. Furthermore this permits shipping of FNA samples at room temperature from peripheral centers to a centralized array core facility.
Rossing
Maria
M
Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark. maria.rossing@rh.regionh.dk
Kaczkowski
Bogumil
B
Futoma-Kazmierczak
Ewa
E
Glud
Martin
M
Klausen
Mikkel
M
Faber
Jens
J
Nygaard
Birte
B
Kiss
Katalin
K
Sørensen
Christian H
CH
Nielsen
Finn C
FC
Bennedbæk
Finn N
FN
Friis-Hansen
Lennart
L
D044963:Biopsy, Fine-Needle; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015203:Reproducibility of Results; D013961:Thyroid Gland / Q000378:metabolism* / Q000473:pathology*
D035683:MicroRNAs