The Luna® Cell Ready One-Step RT-qPCR Kit provides all the necessary components for direct, dye-based RNA detection and quantitation, bypassing the need for RNA extraction and purification. The Luna Cell Ready One-Step RT-qPCR Kit is composed of: 1) the Luna Cell Ready Lysis Module (NEB #E3032S) and 2) the Luna Universal One-Step RT-qPCR Kit (NEB #E3005L).
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 One-Step RT-qPCR Kit is compatible with dye-based real-time quantitation of target RNA sequences via the SYBR®/FAM fluorescence channel of most real-time instruments. In the Luna Universal 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 One-Step RT-qPCR Kit can detect and quantitate RNA transcripts in the cell lysate prepared directly from the Luna Cell Ready Lysis Module.
This table below 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
Figure 1: Luna Cell Ready One-Step RT-qPCR Workflow
The Luna Cell Ready One-Step RT-qPCR Kit provides all the necessary components for direct RNA detection and quantitation from cultured cells (up to 100,000 cells per 50 µl lysis reaction). By coordinating the actions of DNase I and the Luna Cell Ready Protease, the Luna Cell Ready Lysis Module offers a simple workflow resulting in effective cell lysis, RNA release and genomic DNA removal simultaneously in a 15-minute protocol. Up to 2 µl lysate (equivalent to RNA from 0.2 - 4,000 cells) can be transferred into 20 µl downstream RT-qPCR reactions.
Figure 2: The Luna Cell Ready One-Step RT-qPCR Kit offers sensitive and accurate quantitation of RNA directly from cell lysates across 5-log cell inputs
Serial dilutions of HeLa cells (100,000 – 10) were lysed in 50 μl Luna Cell Ready lysis reactions (NEB #E3032) using standard reaction conditions (10 min lysis at 37°C, 5 min inactivation at 25°C). Genes of interest (GOI) were then quantitated using the Luna Universal One-Step RT-qPCR Kit (NEB #E3005) using 1 μl of cell lysate as input (equal to 0.2 – 2,000 cells in a 20 μl RT-qPCR reaction), with duplicate reactions at each input concentration. Results for β-actin, an abundant target (A) and SMG1, a less abundant target (B) are shown. Efficiency (E) and linearity (R2) are shown in right panels.
Figure 3: The Luna Cell Ready One-Step RT-qPCR Kit offers reliable and precise RNA quantitation comparable to purified RNA across 5-log cell inputs
Serial dilutions of A549 cells (100,000 - 10) were lysed in 50 µl Luna Cell Ready lysis reactions (NEB #E3032) using standard reaction conditions (10 min lysis at 37°C, 5 min inactivation at 25°C). Alternatively, RNA was purified using a column-based RNA extraction kit. A. Genes of interest (GOI) were then quantitated using the Luna Universal One-Step RT-qPCR Kit (NEB #E3005) with 1 μl of cell lysate (closed circles) or purified RNA (empty circles) as input (equal to 0.2 – 2,000 cells in a 20 µl RT-qPCR reaction), with duplicate reactions at each input concentration. Left: Detection of β-actin, an abundant target, and ARF3 and Tubulin, two less abundant targets, across 5-logs of cell inputs. Efficiency (E) for each target is shown at the lower left corners of the panel. B. Cq relative to β-actin at each dilution (ΔCq (GOI)) was calculated from the data in (A) across 5-log cell inputs. The average Cqs are shown as bars.
Figure 4: The Luna Cell Ready One-Step RT-qPCR kit outperforms commercially available cell lysis One-Step RT-qPCR kits with respect to both efficiency and sensitivity.
Serial dilutions of A549 cells (100,000 - 10) were lysed in 50 µl Luna Cell Ready lysis reactions (NEB #E3032) using standard reaction conditions. Alternatively, the same amount of cells were lysed with commercially available kits from Bio-Rad (SingleShot SYBR Green One-Step Kit, #172-5095), Qiagen (FastLane Cell SYBR Green Kit, #216213) and Thermo Fisher (Cells-to-CT 1-Step PowerSYBR Green Kit, A25600) following manufacturer-recommended protocols. Genes of interest were then quantitated using the One-Step RT-qPCR module from each kit with 1 μl of cell lysate as input (equal to 0.2 -2,000 cells in a 20 µl RT-qPCR reaction), with duplicate reactions at each input concentration. Amplification of β-actin, an abundant target, and RPL32 and Tubulin, two less abundant targets, across 5-logs of cell inputs are shown for all kits. To standardize results, 12% of total fluorescence was set as a threshold. Efficiency (E) and the Cq of the highest and lowest input are shown.
Figure 5: A set of 24 targets demonstrates the most sensitive quantitation from the Luna Cell Ready One-Step RT-qPCR Kit compared to the commercially available cell lysis One-Step RT-qPCR kits.
Approximately 2,500 A549 cells were lysed in 50 µl Luna Cell Ready lysis reactions (NEB #E3032) using standard reaction conditions or with commercially available kits following manufacturer-recommended protocols. Two biological replicates were processed for each kit. 24 genes of interest were then quantitated using the One-Step RT-qPCR module from each kit with 1 μl of cell lysate as input (equal to 50 cells in a 20 µl RT-qPCR reaction), with duplicate reactions for each biological sample. Average Cqs are shown for NEB (closed orange circles), BioRad (open squares), Qiagen (open triangles) and Thermo Fisher Scientific (crosses). To standardize results, 12% of total fluorescence was set as a threshold. The Luna Cell Ready One-Step RT-qPCR Kit shows the earliest Cq for 23/24 genes across variable expression levels, with an average of 2.3 Cq faster than BioRad, 3.8 Cq faster than Qiagen, and 3.6 Cq faster than Thermo Fisher Scientific.
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Nucleic acid-based aptamers for use with thermophilic DNA polymerases and reverse transcriptases are licensed exclusively by New England Biolabs, Inc. from SomaLogic, Inc. New England Biolabs, Inc. gives the Buyer/User a non-exclusive license to use the Luna® Cell Ready One-Step RT-qPCR Kit for Research Use Only (RUO). Commercial use of this product may require a license from New England Biolabs, Inc. For additional information or to inquire about commercial use, please contact busdev@neb.com.
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