Small-sample total RNA purification: laser capture microdissection and cultured cell applications.
Gene expression studies require analysis of RNA, but isolation of total RNA from very small samples by traditional methods can be difficult and inefficient. The Absolutely RNA microprep kit provides a convenient method for isolating total RNA from small numbers of cells such as those harvested by laser capture microdissection (LCM). The protocol includes binding of RNA to a solid support, thus eliminating the need for organic extraction and alcohol precipitation. DNase digestion on the solid support reduces or eliminates DNA contamination and minimizes RNA handling. Efficient washing removes contaminants, and elution in a small volume of buffer results in high-purity RNA at a concentration appropriate for demanding applications such as RT-PCR. RNA isolated from as few as 200 laser capture microdissected brain tumor cells resulted in detection of low, medium, and highly expressed genes by conventional and real-time RT-PCR.
Dolter
K E
KE
Stratagene, La Jolla, CA, USA.
Braman
J C
JC
D016475:3T3 Cells / Q000737:chemistry; D000818:Animals; D001932:Brain Neoplasms / Q000737:chemistry / Q000473:pathology; D016466:CHO Cells / Q000737:chemistry; D002478:Cells, Cultured / Q000737:chemistry*; D003199:Computer Systems; D006224:Cricetinae; D003412:Cricetulus; D004587:Electrophoresis, Agar Gel; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D018922:HL-60 Cells / Q000737:chemistry; D006367:HeLa Cells / Q000737:chemistry; D006801:Humans; D007834:Lasers; D051379:Mice; D008832:Microchemistry / Q000379:methods*; D008867:Microtomy / Q000295:instrumentation / Q000379:methods*; D009000:Monocytes / Q000737:chemistry; D009361:Neoplasm Invasiveness; D012313:RNA / Q000302:isolation & purification*; D012334:RNA, Neoplasm / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D012334:RNA, Neoplasm; D012313:RNA