Go direct from cells to RNA quantitation without purification
The Luna Cell Ready Probe One-Step RT-qPCR Kit provides all the necessary components for direct, probe-based RNA detection and quantitation, bypassing the need for RNA extraction and purification. The Luna Cell Ready Probe One-Step RT-qPCR Kit contains two modules: 1) the Luna Cell Ready Lysis Module (NEB #E3032S) and 2) the Luna Universal Probe One-Step RT-qPCR Kit (NEB #E3006L).
Cell cultures are often analyzed for gene expression or treatment responses as a proxy for a living organism. Traditionally, RNA is extracted and purified from treated cells via column-based or chemical methods. Coordinating the actions of DNase I and the Luna Cell Ready Protease, the Luna Cell Ready Lysis Module offers a simple alternative workflow resulting in effective cell lysis, RNA release, and genomic DNA removal simultaneously in a 15-minute protocol. The Lysis Module includes a unique Luna Cell Ready RNA Protection Reagent that maintains RNA integrity during cell lysis. The lysis capacity spans 10 -100,000 cells in a 50 µl lysis reaction. Up to 2 µl of lysate (equivalent to RNA from 0.2 - 4,000 cells) can be transferred into 20 µl downstream RT-qPCR reactions. Similar to other Luna products, the lysis buffer includes an inert blue tracking dye for visual assistance throughout the workflow.
The Luna Universal Probe One-Step RT-qPCR Kit is compatible with probe-based real-time quantitation of target RNA sequences via multiple fluorescence channels of most real-time instruments. In the Luna Universal Probe One-Step RT-qPCR Kit, Hot Start Taq DNA polymerase is combined with a novel Luna WarmStart Reverse Transcriptase, allowing dual control of enzyme activity via reversible, aptamer-based inhibition. The Luna Universal Probe One-Step RT-qPCR Kit can detect and quantitate RNA transcripts (via a singleplex or multiplex reaction) in the cell lysate prepared directly from the Luna Cell Ready Lysis Module.
This table lists cells lines that are compatible with the Luna Cell Ready Lysis Module. It includes results seen using a 5-log dilution series (100,000 - 10 cells per 50 µl lysis reaction) and subsequent detection of 1 µl of lysate in a 20 µl one-step RT-qPCR reaction. For each cell line, linear results are indicated in the final column. In addition, some insect cell lines have been tested and are compatible.
Table 1: Validated Cell Lines
|Cell line||Property||Species||Cell number per 50 μl lysate|
|A549||Adherent||H. sapiens, Lung, carcinoma||10 - 100,000|
|HEK293||Adherent||H. sapiens, Kidney||10 - 100,000|
|HeLa||Adherent||H. sapiens, Cervix, adenocarcinoma||10 - 100,000|
|HepG2||Adherent||H. sapiens, Liver, carcinoma||10 - 1,000|
|NCI-H460||Adherent||H. sapiens, Lung, carcinoma||10 - 100,000|
|SK-N-SH||Adherent||H. sapiens, Brain, Neuroblastoma||10 - 10,000|
|U2Os||Adherent||H. sapiens, Bone, osteosarcoma||10 - 100,000|
|Jurkat||Suspension||H. sapiens, T lymphocyte, leukemia||10 - 100,000|
|K-562||Suspension||H. sapiens, Lymphoblast, leukemia||10 - 100,000|
The following reagents are supplied with this product:
|Store at (°C)||Concentration|
|Luna Cell Ready Lysis Buffer||-20||2 X|
|Luna Cell Ready RNA Protection Reagent||-20||25 X|
|Luna Cell Ready Protease||-20||25 X|
|Luna Cell Ready Stop Solution||-20||10 X|
DNase I (RNase-free)
|Luna® WarmStart® RT Enzyme Mix||-20||20 X|
|Luna® Universal Probe One-Step Reaction Mix||-20||2 X|
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