I need a method to measure gene expression by qPCR in very small samples, ideally cells grown in 96-well plates. What method should I use? Any suggestion will be welcome!
Http://www.qiagen.com/products/rnastabilizationpurification/rneasysystem/rneasy96.aspx
there are lots of practical difficulties in doing that and risks of contamination ,better use Eppendorf tubes
We use cell-to-cell lysis buffer (Ambion, LifeTechnologies), a very good solution to easily and rapidly! extract RNA from you 96-well samples. Good luck with it!
We used Triton X100.
Reference: Ranheim T, Mathis PK, Joelsson DB, Smith ME, Campbell KM, Lucas G, Barmat S, Melissen E, Benz R, Lewis JA, Chen J, Schofield T, Sitrin RD, Hennessey JP Jr. Development and application of a quantitative RT-PCR potency assay for a pentavalent rotavirus vaccine (RotaTeq). Journal of Virological Methods 2006 Feb;131(2):193-201. Epub 2005 Oct 7
I use cells to ct Kit to make the cDNA direct from cells (Ambion)...It works really well, very good quality of cDNA, very good amplification in qPCR, almost no variation in efficiency between replicates and comparing to other methods of extraction (colums or trizol) in 96 well plates, this is the best I've ever used... I truly recommend when extracting RNA for qPCR from cells in a 96-well plate.
I agree with Lotte and Ana.cells to cDNA kit is really good in terms of quality and comparisson between samples (very tight 18S values between samples).Is a bit expensive to buy at first but saves money in the end as you don't need to do an RNA extraction and use less reagents.
Julie, Ana and Lotte, thank you for your answers. Do I need to calculate the number of the cells first or measure RNA concentration before qPCR if I use this kit? How does it work?
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