Improved high-throughput ultrafiltration process enables cRNA purification for gene expression profiling.
Ultrafiltration of nucleic acids has been used for a wide variety of applications, including sequence reaction purification and amplicon cleanup prior to spotting onto microarrays. Here we describe a novel process, using ultrafiltration, that purifies cRNA products for sensitive downstream applications. Initial attempts at this high-throughput purification for cRNA resulted in low sensitivity when compared against an industry standard (silica-based bind, wash, and elute purification). We modified the ultrafiltration process to include a proteinase K preincubation and a phosphate buffer wash that, when combined, increased sensitivity and signal-to-noise ratio in microarray applications. The protocol that we have developed eliminates the use of chaotropic salts (such as guanidinium thiocyanate) that are typically used in silica binding purification methods. The data demonstrate good performance for sensitive RNA applications using well-defined metrics, and thus the technique might be useful for a broader range of nucleic acid purifications.
Morris
Mark D
MD
Rosetta Inpharmatics LLC, Seattle, WA 98109, USA. mark_morris@merck.com
Meyer
Michael R
MR
Painchaud
Gary
G
Marlowe
Jon
J
Tokiwa
George Y
GY
Fare
Thomas L
TL
D020869:Gene Expression Profiling; D006801:Humans; D019169:Jurkat Cells; D020014:K562 Cells; D018075:RNA, Complementary / Q000302:isolation & purification*; D014158:Transcription, Genetic; D014462:Ultrafiltration / Q000379:methods*
D018075:RNA, Complementary