Product Class: Kit

Luna® Cell Ready One-Step RT-qPCR Kit 


Catalog #E3030

Product Introduction

Go direct from cells to RNA quantitation without purification

  • Contains reagents for 100 lysis preps and 500 one-step RT-qPCR reactions (dye)
  • Effective cell lysis preparation from 10 to 100,000 cells across numerous cell lines
  • Coordinated cell lysis, RNA release, and genomic DNA removal in a fast 15-minute protocol
  • Features Luna WarmStart® RT paired with Hot Start Taq for increased thermostability and room temperature setup
  • Additional lysis preps available: Luna Cell Ready Lysis Module (NEB #E3032)
 

Product Information

Description

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. 


This product is related to the following categories:
Luna® qPCR & RT-qPCR Products,
PCR, qPCR & Amplification Technologies Products,
This product can be used in the following applications:
qPCR & RT-qPCR,
DNA Amplification, PCR & qPCR

Properties & Usage

Materials Required but not Supplied

  • Phosphate-buffered saline (PBS)
  • Target-specific primers
  • Cells
  • Eppendorf tubes, PCR strip tubes
  • PCR plates
  • qPCR instrument
  • Pipettors and pipette tips (to minimize cross contamination, filter tips should be used)

Protocols, Manuals & Usage

Protocols

  1. Protocol for Luna® Cell Ready One-Step RT-qPCR Kit Protocol (NEB #E3030)

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Application Notes

Tools & Resources

Web Tools

FAQs & Troubleshooting

FAQs

  1. What cell lines are compatible with the Luna Cell Ready One-Step RT-qPCR Kit (NEB #E3030)?
  2. How many cells should I use for reliable detection?
  3. How many cells should I seed into cell culture plates to achieve optimal detection?
  4. Can cryopreserved cell lines or pelleted cells stored at -80°C be used with this kit? 
  5. How can lysis efficiency be determined?
  6. Why is it important to evaluate RNA damage or inhibition during cell lysis and one-step RT-qPCR detection?
  7. What is the “Luna Cell Ready RNA Protection Reagent” and when should it be used?
  8. Should I include a No-RT control reaction?
  9. Will the blue tracking dye interfere with detection?
  10. Is cell washing prior to lysis necessary?
  11. Which lysis format should I use?
  12. What protocol should be used for cell lysis? Can cells be lysed at room temperature or using a short protocol?
  13. When using cell lysates generated from Luna Cell Ready Lysis Module, can RT-qPCR experiments be set up at room temperature?
  14. How much cell lysate should I use as input? (NEB #3030)
  15. Can I use the cell lysates generated from the Luna Cell Ready Lysis Module for two-step RT-qPCR? 
  16. Why does the Luna Cell Ready One-Step RT-qPCR Kit contain more RT-qPCR reactions than lysis reactions?
  17. If either the Luna Cell Ready Lysis Module or Luna Universal One-Step RT-qPCR Kit runs out, can I order them separately?
  18. How can I obtain more technical help related to Luna Universal One-Step RT-qPCR Kit?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Licenses

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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