New England Biolabs, E3030S, Luna® Cell Ready One-Step RT-qPCR Kit

Go direct from cells to RNA quantitation without purification Related Categories Luna® qPCR & RT-qPCR,, PCR, qPCR & Amplification Technologies Applications qPCR & RT-qPCR,, Two-Step RT-qPCR,, DNA Amplification, PCR & qPCR Specification 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) FAQ Q: What cell lines are compatible with the Luna Cell Ready One-Step RT-qPCR Kit (NEB #E3030)? A: Numerous cell lines can be used for direct RNA detection using the Luna Cell Ready One-Step RT-qPCR Kit. However, some cell lines may contain high levels of inhibitors that may affect cell lysis or the RT-qPCR reaction. Culture medium and drug/RNAi/CRISPR treatment can also affect cell growth status and levels of inhibitors. The table below highlights the results observed with various mammalian cell lines using a 5-log dilution series (100,000 -10 cells per 50 µl lysis), and subsequent detection of 1 µl of lysate in a 20 µl one-step RT-qPCR experiment. For each cell line, linear results are indicated in the final column of the table below. In addition to these mammalian cell lines, the kit is also compatible with insect cell lines. For cell lines not listed below, it is recommended to perform a pilot test for the optimal cell input numbers for lysis as well as the amount of lysate that can be carried into RT-qPCR. Cell lines Property Species Cell number per 50 μl lysate HeLa Adherent Hs. Cervix, adenocarcinoma 10 - 100,000 A549 Adherent Hs. Lung, carcinoma 10 - 100,000 Jurkat Suspension Hs. T lymphocyte, leukemia 10 - 100,000 HEK293 Adherent Hs. Kidney 10 - 100,000 U2Os Adherent Hs. Bone, osteosarcoma 10 - 100,000 HepG2 Adherent Hs. Liver, carcinoma 10 - 1,000 K-562 Suspension Hs. Lymphoblast, leukemia 10 - 100,000 NCI-H460 Adherent Hs. Lung, carcinoma 10 - 100,000 SK-N-SH Adherent Hs. Brain, Neuroblastoma 10 - 10,000 Q: How many cells should I use for reliable detection? A: The Luna Cell Ready Lysis Module has a lysis capacity of 10 - 100,000 cells in a 50 µl lysis reaction. Typically, 1-2 µl cell lysate (equivalent to RNA from 0.2 - 4,000 cells) can be transferred into a 20 µl downstream RT-qPCR. Most transcripts can be detected from 20 to 200 cells in a typical 20 µl reaction. However, some cell lines may contain high levels of inhibitors to cell lysis or RT-qPCR reactions. For a quick check of gene expression, ~1000 cells per 50 μl reaction is a good starting point for optimization. Please see additional FAQ’s for guidance regarding specific cell lines. Precious samples with few cells (≤10) can also be lysed with the Luna Cell Ready Lysis Module with subsequent detection using the Luna Universal Probe RT-qPCR Kit. However, modification of the Cell Lysis Mix is required as indicated below. Pilot experiments are recommended to optimize the entire workflow before using precious/limited samples. Components Total volume (25 µL) Luna Cell Ready Lysis Buffer (2X) 12.5 µL DNase I (RNase-free) (10X) 2.5 µL Luna Cell Ready RNA Protection Reagent 2.5 µL Luna Cell Ready Protease 0.25 µL Luna Cell Ready Stop Solution 2.5 µL Cells Variable, up to 2.5 µL RNase-free Water To a final volume of 25 µL Q: How many cells should I seed into cell culture plates to achieve optimal detection? A: Following general cell culture guidelines, do not seed cells to exceed average yields per well at 100% confluence (refer to manufactories’ guidelines, the table below is modified from Corning Inc. as an example). Keep in mind, cells might still be dividing after seeding. Make sure the cell number at harvest is within the lysis capacity. In addition, an initial test may help determine the optimal range of cell inputs for your specific cell lines or amplicons. Cell culture plates Average Yields (100% confluence) 6-wells 9.5 x 105 12-wells 3.8 x 105 24-wells 1.9 x 105 48-wells 0.95 x 105 96-wells 3.2 x 104 384-wells 5.6 x 103 Q: Can cryopreserved cell lines or pelleted cells stored at -80°C be used with this kit? A: Yes, the Luna Cell Ready Lysis Module is compatible with cryopreserved cells provided that the cell viability has been maintained. If frozen cell pellets will be routinely used, aliquot the desired number of cells before pelleting and freezing down, if possible. Q: How can lysis efficiency be determined? A: To determine lysis efficiency, RT-qPCR should be evaluated across a 5-log dilution of cells. Resuspend cells in Cell Resuspension Solution and perform lysis of 10 – 100,000 cells per 50 µl reaction. Perform one-step RT-qPCR with the cell lysates and check the RT-qPCR efficiency using a gene of interest (target/primers should be validated using pure RNA first). The ideal efficiency should be 90%-110%. Efficiency <90% often indicates RNA damage. Efficiency >110% with delayed initial Cq may imply potential inhibitors present in the RT-qPCR. Q: Why is it important to evaluate RNA damage or inhibition during cell lysis and one-step RT-qPCR detection? A: At high concentrations, certain cellular components may damage RNA or inhibit cell lysis and RT-qPCR steps, leading to inaccurate quantitation of RNA expression. In the presence of the Luna Cell Ready RNA Protection Reagent, RNA damage can be dramatically reduced. Two approaches can be used to evaluate proper performance of the Luna Cell Ready workflow. 1) Spike in a reference RNA when preparing your cell lysis mix. (Compare the Cq values of your reference RNA in the presence or absence of cells. Similar Cq values (≤1 Cq difference) indicate limited RNA damage and minimal inhibition to RT-qPCR reactions. It is recommended to use a reference RNA without homology to the RNA population. For example, if you are working with human cell lines, in vitro transcribed Lambda phage RNA may be an option.) 2) Perform one-step RT-qPCR with purified total RNA approximately equivalent to the amount of RNA in the cell lysate. For example, when the cells are harvested, half of the amount can be used for RNA extraction using a column-based kit (e.g., Monarch Total RNA Miniprep Kit, NEB#T2010S) while the other half can be prepared with the Luna Cell Ready Lysis Module. Similar Cq values suggest efficient cell lysis. Otherwise, decrease the cell number until the similar results are achieved. Q: What is the “Luna Cell Ready RNA Protection Reagent” and when should it be used? A: During cell lysis, endogenous nucleases that can nonspecifically degrade cellular RNA are released. The Luna Cell Ready RNA Protection Reagent has been optimized to limit RNA degradation during the cell lysis procedure. We recommend including the Luna Cell Ready RNA Protection Reagent in cell resuspension, dilution and lysis (as specified in the manual) to minimize RNA damage. Q: Should I include a No-RT control reaction? A: No-RT control reactions are useful for determining issues that may arise from the amplification of gDNA that may be present in a sample. In general, the Luna Cell Ready One-Step RT-qPCR Kit offers efficient genomic DNA removal (>95%). For example, with an abundant transcript such as β-actin, genomic DNA is detected approximately 10 cycles later than from RNA detection after DNA removal. However, many RNA transcripts are present at low abundance. For these transcripts, it is recommended to perform a No-RT control reaction, particularly if primer sets do not span exon-exon junctions. In addition, no template control (NTC) reactions should be included to demonstrate that positive reactions are meaningful. Q: Will the blue tracking dye interfere with detection? A: The Luna Cell Ready Lysis Buffer contains an inert visual reference dye. This dye makes it easier to monitor reaction setup and ensure appropriate liquid transfer between lysis and RT-qPCR plates. The blue dye has been extensively evaluated in the Cell Lysis Reaction and RT-qPCR experiments; no inhibition or interference has been observed. Q: Is cell washing prior to lysis necessary? A: For best results, we recommend washing cells to get rid of unwanted media and dead cells. However, in situations where this is not practical, such as high throughput screening or extremely sensitive gene expression studies where cell washes may change RNA abundance, plate flipping can be used for effective medium removal. If the washing step is omitted, lysates should be used immediately, without freeze and thaw cycles. Q: Which lysis format should I use? A: For adherent cells growing in plates, lysis can be performed in the plate to simplify the workflow and reduce cell loss. For non-adherent cells, suspend cells in Cell Resuspension Solution and lyse an appropriate volume (see protocol for details). Q: What protocol should be used for cell lysis? Can cells be lysed at room temperature or using a short protocol? A: For best results, we recommend using a standard lysis protocol of 10 min at 37°C followed by inactivation for 5 min at 25°C. However, lysis temperatures from 25-37°C have been tested with the Luna Cell Ready Lysis Module. If the cell number is less than 10,000 (per 50 µl lysis reaction), 10 min at 25°C can also be used. Although the recommended period is 10 min at 37°C for cell lysis, 5 -20 min incubations have also been tested. Incubation at 37°C for 5 min is acceptable for RNA release if genomic DNA removal is NOT a high priority. Please note that the subsequent inactivation step (5 min) should not be reduced. Use the lysate immediately or save on ice and use within 5 hours. Q: When using cell lysates generated from Luna Cell Ready Lysis Module, can RT-qPCR experiments be set up at room temperature? A: Yes, RT-qPCR can be set up at room temperature. However, cell lysates are sensitive to temperature; if possible, keep the lysates on ice during reaction setup. Once the RT-qPCR plate is set up, run the reactions as soon as possible. If short term storage of the RT-qPCR plate is required, keep at 4°C for no longer than 5 hours for dye-based detection. For probe-based detection, RT-qPCR experiment should proceed immediately after setup. For high throughput screening using automation, minimize the time that lysates sit at room temperature (no longer than 15 min prior to starting process), and avoid freeze and thaw cycles. Q: How much cell lysate should I use as input with Luna Cell Ready One-Step RT-PCR? A: The amount of cell lysate that can be added to one-step RT-qPCR will depend on the RT-qPCR kit. When using the Luna Universal One-Step RT-qPCR Kit, lysate can easily comprise up to 10% of total reaction volume and ≤ 4,000-cells per 20 μl RT-qPCR experiment. Q: Can I use the cell lysates generated from the Luna Cell Ready Lysis Module for two-step RT-qPCR? A: For best results, we recommend using the lysates in one-step RT-qPCR. If cell lysates are planned for two-step RT-qPCR, pilot experiments are recommended to optimize cell lysate inputs. Approximately 200 cells per 20 μl RT reaction is a good starting point for optimization. Q: Why does the Luna Cell Ready One-Step RT-qPCR Kit contain more RT-qPCR reactions than lysis reactions? A: The Luna Cell Ready One-Step RT-qPCR Kit pairs a lysis module containing 100 lysis reactions with a One-Step RT-qPCR module of 500 reactions. This pairing is based on a typical experimental setup where a user may run multiple replicates of a transcript of interest (plus a reference) with appropriate no-RT controls. Conservatively, a 50 µl cell lysis reaction is sufficient at least for 40 RT-qPCR reactions assuming 1 µl lysate per reaction. Scale up lysis reactions accordingly if more RT-qPCR reactions are planned. Both the Luna Cell Ready Lysis Module (NEB #E3032) and the Luna Universal One-Step RT-qPCR Kit (NEB #E3005) can be ordered separately if necessary. Q: If either the Luna Cell Ready Lysis Module or Luna Universal One-Step RT-qPCR Kit runs out, can I order them separately? A: Yes. Both modules can be ordered separately. The Luna Cell Ready Lysis Module (NEB #E3032) is available as 100 lysis reactions (50 µl). The Luna Universal One-Step RT-qPCR Kit (NEB #E3005) has four sizes (20 µl per reaction), E3005S (200 reactions), E3005L (reactions), E3005X (1,000 reactions) and E3005E (2,500 reactions). The overall performance of one-step cell direct RT-qPCR depends both on the lysis module and the One-Step RT-qPCR kit. For best results, it is recommended to pair both modules together. Q: How can I obtain more technical help related to Luna Universal One-Step RT-qPCR Kit? A: Please refer to additional FAQ and manual located at www.neb.com/E3005 or www.neb.com/E3006.