SLAS 2014: In the video above, Dr. Rupal Cutting explains how Roche’s new high-throughput, completely automatable qPCR workflow easily surpasses reporter gene assays.
Taken from LabCyte White Paper, “High Throughput RT-qPCR for Gene Expression Analysis.”
Unlike reporter gene assays, RT-qPCR can be used on native, non-transformed cell types, and RT-qPCR measures mRNA transcripts of the actual gene as it is expressed from within its natural environment, rather than transcripts from an artifial, plasmid-based consrtuct.*
Cited Papers Referencing Known Issues:
Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression.
Douglas S. Auld, Natasha Thome, William F. Maguire, and James Inglese. NIH Chemical Genomics Center, National Institutes of Health, Bethesda, MA 20892-3370
False Positives in a Reporter Gene Assay: Identification and Synthesis of Substitues N-Pyridin-2-yl-benzamides as Competitive Inhibitors of Firefly Luciferase.
J Med Chem, 2008, 51, 4727-4729 Heitman, L.H.; Veldhoven, J.P.D.; Zweemer, A.M.; Ye, K.; Brusse, J.; Ijzeman, A.P.
Reporter gene assays
|1||Start with treated cells in a 384- or 1536- well tissue culture plate|
|2||Add lysis buffer, incubate at room temperature for 5 minutes|
|3||Transfer lysates and one-step master mix to qPCR plate|
|4||Seal and centrifuge|
The LightCycler® 1536 optical unit facilitates mono- and dual-color assays using a variety of dyes, such as FAM, VIC, and Yellow 555, by supporting formats including Universal ProbeLibrary (UPL) probes, hydrolysis probes, and intercalating dyes (e.g., BrightGreen). The unique thermal block cycler design, in combination with the LightCycler® 1536 Multiwell Plate, enables very high well-to-well homogeneity and inter-assay reproducibility.
The LightCycler® 1536 Software supports the next generation of real-time PCR results (e.g., Cp's, endpoint fluorescence values), providing novel threshold functions for data refinement and powerful tools for data sorting and reviewing. Since this system is designed for automated high-throughput laboratory workflows, the instrument’s software allows easy data management for network and LIMS environments. In combination with Roche RealTime ready reagents, the system also features a new surveillance concept that checks for pipetting errors.
57.4 cm x 58.8 cm x 49.7 cm
22.6 in x 23.1 in x 19.6 in
(W x D x H)
|Weight||55.6 kg (121 lbs)|
|Computer||PentiumPC with Windows XP|
|Reaction Vessel||LightCycler® 1536 Multiwell Plate|
|Reaction Volume||0.5 – 2.0 µl|
|Excitation Source||Xenon lamp|
Excitation: 465 nm, 533 nm
Detection: 510 nm, 580 nm
|Detection||Cooled monochrome CCD camera|
|Temperature Range||+37 to +95°C|
Therma-BaseTM heat spreading technology
|Run Time||<50 minutes|
Thermaxis® technology (two-component design)
Barcode labeled for automated workflow tracking
The Future of High Throughput Assays Webinar
High Throughput RT-qPCR for Gene Expression Analysis White Paper
Video: High Throughput RT-qPCR for Identifying Activators of Latent HIV-1 in Primary Human CD4+ T Cells
Helen Yu, PhD, Biology Department, Gilead Sciences
Video: Validation of an automation compatible high-throughput Reverse Transcription qPCR workflow
Dr. Catherine Bardelle, AstraZeneca, Alderley Park, Macclesfield, UK