alexa Represe ntative in Vitro cDNA Amplifica tion F rom Ind ividu al Hemopo ietic Cells and Colon ies


Single Cell Biology


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A simple procedu re is des cribed for preparing microgram amoun fs of cDN A, suitable for clon ing into libraries , from samples as s mall as a s ing le ce ll. Syn- thes is of cDN A is primed with o ligo (dT ) and the res ulting strand s a re tailed with po ly(dA). Th e c DNA is then amplified u sing an o ligo(dT)- con taining se quence to p rime the po lymerase c hain reaction . By limiting the size of the first cDN A strand s, efficient amplification is ac hieved ind ependently of size of the original mRN A transc ripts. The method was app lied to small numbers of hemopo ietic ce lls of variou s lineages , as well as to sing le micromanipu lated cells. Th e res ults es tablished the fidelity and spec ificity of t he a mplified p rodu ct, and showed that se nsitivity was s ufficient t o d etec t mod erate-to-low abu ndance mess ages in a s ingle cell. Amplifica tion o f defined RNA mixtures c on firmed that the proce dure c ou ld d etec t low abund ance s equ ences a t least as rare as .02 5%, and that relative a bund ance was prese rved du ring amplifica tion .

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This article was published in METHODS IN MOLECULAR AND C ELL ULAR B IOLOGY and referenced in Single Cell Biology

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