RNA was isolated from 1 to 1 x 105 HeLa S3 cells using the RNeasy 96 BioRobot 8000 procedure. Total RNA was eluted in 100 µl RNase-free water, and 5 µl was used for RT-PCR. Quantitative, real-time one-step RT-PCR analysis was carried out on an ABI Sequence Detection System using the QuantiTect Probe RT-PCR Kit with primers and probe specific for the low-copy c-fos transcript. [A] Amplification plot. [B] CT values. Error bars represent standard deviation from 8 different samples for each cell number.
[A] Agarose gel analysis. HeLa cells (5 x 105) from a homogeneous cell culture were pelleted in each well of a 96-well cell-culture plate and the total RNA isolated using the RNeasy 96 system. Half of each eluate was analyzed on a formaldehyde agarose gel. [B] The corresponding northern blot, hybridized with a 32P-labeled GAPDH probe. [C] Analysis of RNA yields. Each square represents the yield from a preparation originating from one well of the 96-well cell-culture plate.
RT-PCR of cytoplasmic RNA isolated with the RNeasy 96 Kit from the indicated numbers of HeLa cells. 10 µl (1/10) of the eluate was reverse-transcribed with oligo-dT primer, and 5 µl (1/5) of the cDNA mix was used in a 100 µl PCR in which a 214 bp fragment of beta-actin was amplified. C-: no template; C+: beta-actin in vitro transcript; M: 100 bp DNA ladder.
RNA was isolated from 96 aliquots (5 x 104 cells each) of a HeLa S3 cell culture using the RNeasy 96 system and the RNeasy 96 BioRobot 8000 procedure. Quantitative, real-time one-step RT-PCR was set up in the same protocol on the BioRobot 8000 workstation, using the QuantiTect Probe RT-PCR Kit with primers and dual-labeled probe specific for the low-copy c-myc transcript. Threshold cycles (CT) are shown for all 96 samples. The mean CT was 21.34 ± 0.34 (mean ± standard deviation), representing a CV of 1.6%.