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Procedure	Time(min)
Prepare Cell Lysis Reagent	5
Wash cells with cold PBS	5
Add Cell Lysis Reagent and incubate	5
Total Time for Cell Lysis	15
Reverse Transcription Setup	15
Reverse Transcription	45
PCR Setup	15
Real-Time PCR with the Universal ProbeLibrary	45
Total Time from Lysis to Real-Time PCR Results	135

", "Country": "XG", "Code": "Assay Time", "Name": "Assay Time" }, { "Language": "en", "Value": "The RealTime ready Cell Lysis Kit is designed for rapid, high-performance gene expression studies using cultured cells in two-step real-time RT-PCR applications. The kit uses an advanced one-step protocol for lysing 3 to 30,000 cells within 5 minutes at room temperature. The resulting lysate can be used directly in cDNA synthesis, eliminating the time-consuming RNA purification step. The cDNA can be analyzed in real-time PCR experiments at any throughput (e.g., LightCycler^® 480 Instrument, 96- and 384-well; LightCycler^® 1536 Instrument, 1536-well).
Due to the user-friendly packaging and easy-to-use protocol, the RealTime ready Cell Lysis Kit can be easily adapted to all experimental and throughput requirements. In addition, the kit easily integrates into all manual and automated cell-based gene expression workflows.", "Country": "XG", "Code": "Applications", "Name": "Applications" }, { "Language": "en", "Value": "To obtain optimal results in downstream procedures, especially in real-time RT-PCR assays on the LightCycler^® Instruments, do not process samples larger than this kit is designed to handle.
The optimal amount of sample material is as follows:

• Seed an appropriate number of adherent cells and grow them overnight.
• The maximal cell number and the volume of Cell Lysis Reagent depends on the type of culture plate used.
  
  

For a 96-well plate:
3 to 3 × 10⁴ cells per well are recommended. Depending on the cell size, up to 10⁵ cells can be used. Although, higher concentrations may result in loss of signal, as the maximal number of adherent cells is restricted by cell density.

Cell Types that we have tested:
Table 1 lists the adherent cell types that we have tested with the RealTime ready Cell Lysis Kit

Cell Line	Organism	Disease/ Tissue
BT-549	human	breast ductal carcinoma
HeLa	human	cervical adenocarcinoma
HepG2	human	hepatocellular carcinoma
HT-29	human	colorectal adenocarcinoma
HT-1080	human	connective tissue fibrosarcoma
HUV-EC-C	human	vascular endothelium from umbilical vein
MCF7	human	breast adenocarcinoma
MDA-MB-231	human	breast adenocarcinoma
MDA-MB-468	human	breast adenocarcinoma
NIH/3T3	human	embryonic fibroblast
SK-BR-3	human	breast adenocarcinoma

", "Country": "XG", "Code": "Sample Materials", "Name": "Sample Materials" }, { "Language": "en", "Value": "Cell lysates have also been tested using reagents from the LightCycler^® Carousel-Based System.", "Country": "XG", "Code": "Protocols - Help Corner", "Name": "Protocols - Help Corner" }, { "Language": "en", "Value": "The kit is designed for:

• Cat. No. 05 943 523 001: 500 lysis reactions, with a final volume of 40 μl each
• Cat. No. 06 366 821 001: 50 lysis reactions, with a final volume of 40 μl each

Vial/Bottle	Cap	Label	Function / Description	Catalog Number	Content
1	colorless	RealTime ready Lysis Buffer	Easy-to-use Lysis Buffer	05 943 523 001	1 bottle, 20 ml
06 366 821 001	1 bottle, 2 ml
2	colorless	Protector RNase Inhibitor	Contains Protector RNase Inhibitor (40 U/μl) To prevent RNA degradation during lysis of the cells	05 943 523 001	1 vial, 250 μl
06 366 821 001	1 vial, 25 μl
3	colorless	Thermolabile Nuclease	To degrade double-stranded DNA during the Reverse Transcription reaction (optional)	05 943 523 001	1 vial, 250 μl
06 366 821 001	1 vial, 25 μl

", "Country": "XG", "Code": "Content", "Name": "Content" }, { "Language": "en", "Value": "For lot-specific certificates of analysis, see section Contact and Support.", "Country": "XG", "Code": "Quality Control", "Name": "Quality Control" }, { "Language": "en", "Value": "Always run a negative extraction control with the samples. To prepare a negative extraction control, replace the cells with PCR-grade water*, or PBS*. In case of a contamination problem, this can be observed with the negative control.
Also, run a negative (no template) control and a RT negative (no RT) control in the RT-PCR. To prepare a negative (no template) control, replace the cell lysate with PCR-grade water*. To prepare a RT negative (no RT) control, perform PCR of the RNA template, in combination with e.g. LightCycler^® 480 Probes Master*, or RealTime ready DNA Probes Master*. In this experimental setup, the reverse transcription step is omitted, thus any PCR product generated is a signal for DNA contamination of the RNA template preparation.", "Country": "XG", "Code": "Control Reactions", "Name": "Control Reactions" }, { "Language": "en", "Value": "Easy-to-use reagent for lysing cells, prior to reverse transcription.", "Country": "XG", "Code": "Product Characteristics", "Name": "Product Characteristics" }, { "Language": "en", "Value": "How this Product Works
RealTime ready Cell Lysis Kit is an easy-to-use reagent for lysing cultured cells in 96- and 384-well plates, prior to reverse transcription. This product is designed specifically for gene expression assays using the Universal ProbeLibrary and a LightCycler^® Master mix on the LightCycler^® 480 System (96- or 384-well format), LightCycler^® 96 Instrument or LightCycler^® 1536 Instrument.
All that is required are the cells, RT-PCR reagents, primers and Universal ProbeLibrary probe.

Test Principle
The isolation of RNA from experimental samples is the first step in gene expression studies. RNA isolation can be a quite time-consuming and intensive process, especially when dealing with samples with small amounts of material/cells.
The RealTime ready Cell Lysis Kit is designed to quickly lyse cultured cells, prior to reverse transcription, without purifying the RNA, thus ensuring minimal loss of RNA and simplification of the whole gene expression workflow.
The basic steps of using the RealTime ready Cell Lysis Kit and subsequent RT-PCR are:

1. Cultured cells, seeded in a 96-, or 384-well plate are washed with ice-cold PBS.
2. Cells are lysed by the addition of the Cell Lysis Reagent and incubation at room temperature. RNA is released from the lysed cells and protected from degradation, due to the presence of Protector RNase Inhibitor in the Cell Lysis Reagent.
3. Cell lysates are reverse transcribed into cDNA with Transcriptor Reverse Transcription. Genomic DNA from the cell lysate is optionally degraded by the addition of Thermolabile Nuclease (provided in the RealTime ready Cell Lysis Kit) to the reverse transcription reaction and a 10 min incubation at 29°C, prior to the reverse transcription step.
4. cDNA is then amplified using e.g. LightCycler^® 480 Probes Master, or RealTime ready DNA Probes Master and the Universal ProbeLibrary.", "Country": "XG", "Code": "Principle", "Name": "Principle" }, { "Language": "en", "Value": "RealTime ready Cell Lysis Kit is designed for the lysis of cultured cells. Cell lysates can then be directly used for cDNA synthesis, using a reverse transcription kit for cDNA synthesis, followed by an appropriate PCR reagent for use on the LightCycler^® Instruments or standard thermal block cyclers. The quality of RNA isolated is suitable for relative quantification of mRNA by RT-PCR, especially on the LightCycler^® Instruments.

Cell lysates can be processed immediately for RT-PCR, or stored frozen at -15 to -25°C, or stored at +2 to +8°C for a maximum of 2 days.

Cell Lysis Volumes Using 1536 plates
Please refer to the instructions for use for RealTime ready Cell Lysis Buffer (Cat. No. 07248431001): p. 6: For adherent cells in 1536 plates for 2 μl residual media add 1.95 μl Buffer and 0.05 μl Protector.", "Country": "XG", "Code": "Background Information", "Name": "Background Information" }, { "Language": "en", "Value": "

• Standard laboratory equipment
• Pipettes and nuclease free, aerosol-preventative tips
• Sterile reaction tubes (Eppendorf) for preparing the Cell Lysis Reagent, master mixes and dilutions
• Phosphate Buffered Saline (PBS) for washing the cells
   

For Reverse Transcription:

• Transcriptor Reverse Transcriptase*
• Transcriptor First Strand cDNA Synthesis Kit*
• Transcriptor High Fidelity cDNA Synthesis Kit*
• Transcriptor Universal cDNA Master*
• Primer for cDNA Synthesis* or Primer \"random\"*
   

For Two-Step RT-PCR:

• LightCycler^® 480 Probes Master*
• RealTime ready DNA Probes Master*

For One-Step RT-PCR:

• LightCycler^® 480 RNA Master Hydrolysis Probes*

For Gene Expression:

• Universal ProbeLibrary assays^§
• RealTime ready Focus Panels^§
• RealTime ready Custom Single Assays^§
• RealTime ready Custom Panels^§

* available from Roche Diagnostics; see Ordering Information for details. 
^ξ for detailed information, please visit www.universalprobelibrary.com and www.realtimeready.roche.com.", "Country": "XG", "Code": "Additional Equipment and Reagent Required", "Name": "Additional Equipment and Reagent Required" }, { "Language": "en", "Value": "I) Handling Requirements

• Complete each phase of the RT-PCR workflow before proceeding to the next phase. For example, you should finish RT-PCR sample preparation before starting RT-PCR set-up. Sample preparation, RT-PCR set-up and the RT-PCR run itself should also be performed in separate locations.
• Do not pool reagents from different lots or from different bottles of the same lot.
• Do not use a kit after its expiration date has passed.
• Cell Lysis Buffer (bottle 1) contains guanidinium salts, which are irritants. Do not let Cell Lysis Buffer touch your skin, eyes, or mucous membranes. If contact does occur, wash the affected area immediately with large amounts of water. If you spill the reagent, dilute the spill with water before wiping it up.
• Do not allow the Cell Lysis Buffer to mix with sodium hypochlorite (bleach) solution or strong acids. This mixture can produce a highly toxic gas.
   

II) Laboratory Procedures

• Use RNase-free Techniques: RNase contaminated reagents and reaction vessels will degrade template RNA.
• Do not eat, drink or smoke in the laboratory work area.
• Do not pipette by mouth.
• Wear protective disposable gloves, laboratory coats and eye protection, when handling samples and kit reagents. Change disposable gloves frequently.
• Do not contaminate the reagents with bacteria, virus or ribonucleases. Use disposable pipettes and RNase-free pipette tips only, to remove aliquots from reagent bottles. Use the general precautions described in the literature.
• Avoid touching surfaces or materials that could cause RNase carryover.
• Use only the reagents provided in this kit. Substitutions may introduce RNases.
• Clean and decontaminate work areas and instruments, including pipettes, with commercially available decontamination reagents.
• Use only new RNase-free aerosol-blocking pipette tips and microcentrifuge tubes.
• Use a work area specifically designated for RNA work and if possible use reaction vessels and pipettes dedicated only for work with template RNA.
• Regarding precautions for safe handling of RNA, see the Roche Life Science Lab FAQS (https://lifescience.roche.com/labfaqs).
• Wash hands thoroughly, after handling samples and reagents.
   

III) Waste Handling

• Discard unused reagents and waste in accordance with country, federal, state and local regulations.
• Material Safety Data Sheets (MSDS) are available on the Roche Life Science homepage (https://lifescience.roche.com),or upon request from the local Roche office.

IV) For customers in the European Economic Area
Contains SVHC: octyl/nonylphenol ethoxylates. For use in research and under controlled conditions only – acc. to Art. 56.3 and 3.23 REACH Regulation.", "Country": "XG", "Code": "Safety Information", "Name": "Safety Information" }, { "Language": "en", "Value": "Follow this procedure to prepare Cell Lysis Reagent for one well:

1. Thaw the frozen Lysis Buffer (bottle 1) at 37°C, until the solution is clear, then equilibrate on ice.
2. To ensure maximal recovery of all the contents, briefly spin the Protector RNase Inhibitor (vial 2) in a microcentrifuge before opening and mix carefully by pipetting up and down.
3. In a 1.5 ml reaction tube on ice, prepare the Cell Lysis Reagent per well, by adding the following components in the order mentioned below:
   
   

Component	Volume for one well [μl]	Final conc.
RealTime ready Lysis Buffer (bottle 1)	39.5	1x
Protector RNase Inhibitor (vial 2)	0.5	20 U
Total volume	40.0

", "Country": "XG", "Code": "Working Solution", "Name": "Working Solution" } ] } } ] }