Kralj Jason G, Player Audrey, Sedrick Hope, Munson Matthew S, Petersen David, Forry Samuel P, Meltzer Paul, Kawasaki Ernest, Locascio Laurie E
National Institute of Standards and Technology, 100 Bureau Dr MS 8313, Gaithersburg, MD, USA.
Lab Chip. 2009 Apr 7;9(7):917-24. doi: 10.1039/b811714d. Epub 2008 Dec 15.
We have demonstrated in vitro transcription (IVT) of cDNA sequences from purified Jurkat T-cell mRNA immobilized on microfluidic packed beds down to single-cell quantities. The microfluidically amplified antisense-RNA (aRNA) was nearly identical in length and quantity compared with benchtop reactions using the same starting sample quantities. Microarrays were used to characterize the number and population of genes in each sample, allowing comparison of the microfluidic and benchtop processes. For both benchtop and microfluidic assays, we measured the expression of approximately 4000 to 9000 genes for sample amounts ranging from 20 pg to 10 ng (2 to 1000 cell equivalents), corresponding to 41 to 93% of the absolute number of genes detected from a 100 ng total RNA control sample. Concordance of genes detected between methods (benchtop vs. microfluidic) and repeats (microfluidic vs. microfluidic) typically exceeded 90%. Validation of microarray by Real-time PCR of a panel of five genes suggests transcription of genes present is approximately six times more efficient with the microfluidic IVT compared with benchtop processing. Microfluidic IVT introduces no bias to the gene expression profile of the sample and provides more efficient transcription of mRNA sequences present at the single-cell level.
我们已经证明，固定在微流控填充床上的纯化Jurkat T细胞mRNA的cDNA序列能够进行体外转录（IVT），低至单细胞数量。与使用相同起始样品量的台式反应相比，微流控扩增的反义RNA（aRNA）在长度和数量上几乎相同。微阵列用于表征每个样品中基因的数量和群体，从而可以比较微流控和台式方法。对于台式和微流控分析，我们测量了样品量从20 pg到10 ng（2到1000个细胞当量）时约4000至9000个基因的表达，这相当于从100 ng总RNA对照样品中检测到的基因绝对数量的41%至93%。两种方法（台式与微流控）之间以及重复实验（微流控与微流控）之间检测到的基因一致性通常超过90%。通过对一组五个基因进行实时PCR对微阵列进行验证，结果表明，与台式处理相比，微流控IVT对存在的基因转录效率大约高六倍。微流控IVT不会对样品的基因表达谱产生偏差，并且能在单细胞水平上对存在的mRNA序列进行更有效的转录。
