Accurate and precise transcriptional profiles from 50 pg of total RNA or 100 flow-sorted primary lymphocytes.
We have developed a total RNA amplification and labeling strategy for use with Affymetrix GeneChips. Our protocol, which we denote BIIB, employs two rounds of linear T7 amplification followed by Klenow labeling to generate a biotinylated cDNA. In benchmarking studies using a titration of mouse universal total RNA, BIIB outperformed commercially available kits in terms of sensitivity, accuracy, and amplified target length, while providing equivalent results for technical reproducibility. BIIB maintained 50 and 44% present calls from 100 and 50 pg of total RNA, respectively. Inter- and intrasample precision studies indicated that BIIB produces an unbiased and complete expression profile within a range of 5 ng to 50 pg of starting total RNA. From a panel of spiked exogenous transcripts, we established the BIIB linear detection limit to be 20 absolute copies. Additionally, we demonstrate that BIIB is sensitive enough to detect the stochastic events inherent in a highly diluted sample. Using RNA isolated from whole tissues, we further validated BIIB accuracy and precision by comparison of 224 expression ratios generated by quantitative real-time PCR. The utility of our method is ultimately illustrated by the detection of biologically expected trends in a T cell/B cell titration of 100 primary cells flow sorted from a healthy mouse spleen.
Shearstone
Jeffrey R
JR
Research Molecular Discovery, Biogen Idec, Inc., 14 Cambridge Center, Cambridge, MA 02142, USA. jeff.shearstone@biogenidec.com
Allaire
Normand E
NE
Campos-Rivera
Juanita
J
Rao
Sambasiva
S
Perrin
Steven
S
D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D019921:Biotinylation; D002469:Cell Separation; D005434:Flow Cytometry; D051379:Mice; D021141:Nucleic Acid Amplification Techniques / Q000379:methods* / Q000592:standards; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000235:genetics* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013154:Spleen / Q000166:cytology; D013601:T-Lymphocytes / Q000378:metabolism*; D014158:Transcription, Genetic
D012313:RNA