1. One-step RT-qPCR assay without RNA extraction for detecting gene expression levels in cells: HEK293 cells were enzymatically digested, washed with DMEM, and then counted. The cells were distributed into six wells of a 96-well plate, with cell numbers of 15,000, 5,000, 1,667, 550, and 183 per well, respectively. The last well served as a DMEM blank control. After centrifugation to remove the supernatant, cell lysis buffer was added according to the kit instructions for cell lysis, followed by RT-qPCR analysis. Dark blue, light blue, dark green, light green, dark yellow, and light yellow represent the six sample groups mentioned above. A and C show the amplification results from the kit and those from a similar product offered by a well-known domestic vendor, respectively; C and D present their corresponding melting curve diagrams. It is evident that, in terms of both detection sensitivity and specificity, the UA kit outperforms the competing product.
Extraction-free One-Step RT-qPCR Kit（SYBR Green）
Extraction-free One-Step RT-qPCR Kit（SYBR Green）
Product Details
Product Details
Product Specification
| Synonyms | 免提取一步法RT-qPCR检测试剂盒（染料法） |
| Stability & Storage | The one-step RT-qPCR assay kit for direct extraction-free use (dye-based) should be stored at −20°C or lower. After first use, any remaining L reagent and S reagent can be stored at 4°C for one month, or aliquoted and stored long-term at −20°C. The cell lysis L reagent supplemented with DNase I should be freshly prepared as needed and is not recommended for storage. The RT-qPCR Mix should be aliquoted and stored to avoid repeated freeze-thaw cycles. DNase I should also be aliquoted and stored to avoid repeated freeze-thaw cycles. ROX should be aliquoted and stored to avoid repeated freeze-thaw cycles. |
Background
RT-qPCR is widely used for gene expression detection and quantification. Although traditional RT-qPCR offers a one-step method, it involves RNA extraction from samples, making the process complex, subject to numerous influencing factors, and resulting in very low throughput. The EF One-Step RT-qPCR Assay Kit is an extraction-free, one-step RT-qPCR kit: after cell washing, lysis buffer is added directly to lyse the cells, and the lysate is used as the template for RT-qPCR amplification. From cell preparation to the completion of qPCR, the entire procedure can be finished in under two hours. Standardized protocols reduce both the time required and the number of variables, significantly increasing assay throughput.
Components
One-step extraction-free methodRT-qPCRThe components and specifications of the test kit are shown in the following table. For each sample50μLof cell lysis volume and20μL RT-qPCRreaction system,Q1001andQ1002can respectively perform100reactions and1000reactions.
specifications |
Lreagent |
Sreagent |
DNase I |
RT-qPCR Mix |
ROX |
100T |
5 ml |
0.5 ml |
50μl |
0.4 mL |
20μl |
1000T |
5x 10 ml |
5 ml |
500μl |
4x 1ml |
200μ |
Protocol
EF One-Step RT-qPCRFor the dye method (SYBR Green I),to ensure detection specificity and sensitivity,all parameters of RT-qPCRneed to be optimized, especially primer design and cell number are crucial.qPCRThe best primers are located at the junction of adjacent exons; such primers can only amplify usingmRNAas the template; or the primers are located on adjacent exons, and the intron between the exons is relatively long. The amplification efficiency of primers using genomicDNAas the template is much lower than that usingmRNAas the template, so amplification using genomicDNAas the template can be ignored. Primer design should also aim to minimize primer dimers as much as possible. It is recommended to use professional primer design software, such asPrimer 3, IDT PrimerQuest, Primer-BLASTfor primer design, selecting2-3pairs of primers for testing, and choosing the optimal primer pair for experiments.
Cells need to be detected within a range of50to20,000Within a single cell range (50μlcell lysisLreagent, 1-400cells/μl), optimize the amount of cell lysis solution added according to gene expression abundanceRT-qPCRreaction volume, recommended20μlofRT-qPCRreaction system does not exceed400cells,the volume of cell lysis solution does not exceed2μl.
RegardingDNase Itreatment:In many cases, such as pseudogenes, genes without introns, or unknown factors, it becomes impossible to avoid genomicDNAamplification through primer design.DNase Itreatment can degrade genomicDNA,reduce the impact of genomicDNAamplification.The expression level ofmRNA will also determine how much genomicDNAamplification affects the quantification ofmRNA. For highly expressedmRNAthe amount far exceeds the number of genomicDNAgene copies; therefore, there is a difference between products withRTand withoutRTthat is>10 Ct,and due to template competition, in theRT-qPCRreaction, genomicDNAThe amplification probability is much lower than that of high-abundancemRNA, so the impact of genomicDNAamplification on gene quantification can be ignored. For genes with extremely low expression levels,mRNA(each cell contains only a fewmRNAs) in quantity, the number of genomicDNAgene copies is comparable; in such cases, it is necessary to rely onDNase Ito degrade genomicDNA,thereby reducing the influence of genomicDNAamplification.,At this point, the duration of cell lysis/DNase Itreatment can be extended to20 minutesin order to more effectively degrade genomicDNA. When measuring genes in organisms known not to containDNAat certain stages—for example, determining the expression of influenza virus genes within cells after infection—theLreagent does not need to includeDNase I.
1.Cell preparation: Cell detection should be performed within a range of50to20,000cells. For adherent cells (96-well or384-well), after aspirating the culture medium, wash once with pre-cooledPBS, and try to completely remove thePBSbefore proceeding according to step3Add cell lysis buffer. After centrifuging suspended cells to remove the culture medium, wash once with pre-cooledPBS, and try to discard as much of thePBSas possible, then use5μlof pre-cooledPBSResuspend the cells and transfer them to a96-well plate,then, according to step3add the cell lysis buffer
2.Cell lysis buffer plusDNase Iis prepared as follows:DNase Iis at a concentration of100x, 990 μlfor cell lysisLreagent is added10μl DNase I
3.To the cell culture plate prepared in step1(a96-well or384-well) add per well50μlcell lysisLreagent(DNAse I), gently pipette up and down5times, and incubate at room temperaturefor 10minutes. (If necessary, to better degrade genomicDNA, the incubation time can be extended to20minutes.)
4.Add per well in the cell culture plate5μlcell lysis terminationSreagent, gently pipette up and down5times, and incubate at room temperature2minutes. (This cell lysis product can be used as a template forRT-qPCR, and it is recommended to proceed with theRT-qPCRreaction immediately, keeping it on ice for no more than4hours. For short-term storage,-20℃ storage1week; for long-term storage, it can be kept at-80℃)
5.RT-qPCRreaction setup:
It is recommended to assemble theRT-qPCRreaction in a clean bench, and add templates—including cell lysate samples and internal controls—in separate, isolated areas to avoid cross-contamination and aerosol contamination. All experiments should use nuclease-free ultrapure water, pipette tips, and reaction tubes. It is advisable to use filter-tipped pipette tips, especially when adding templates, to prevent cross-contamination from the pipette tip.
It is recommended that RT-qPCR be performed in a20μlreaction system, assembled on ice. The RT-qPCR Mix reagent in the kit is 5x concentrated; according to the table below, add gene-specific primers (final concentration100-400 nM), and the cell lysis product (0.5-2μl, prepared in steps 1-4) and ROX. The amount of ROX should be determined based on the qPCR instrument: for instruments that do not require ROX, add ROX at a high concentration (100x;0.2μl /20μlreaction) or, for instruments requiring low-concentration ROX, add it at 1000x (0.02μl /20μlreaction). For details on major qPCR instruments and ROX concentrations, please refer to the appendix or consult the qPCR instrument's user manual and manufacturer.
20μlRT-qPCR reaction system
RT-qPCR Mix (5x) |
4 μl |
Gene-specific primer pair |
It is recommended that the final concentration of each primer be200 nM |
Cell lysate (template) |
It is recommended0.5–1 μl, not exceeding2 μl, with the number of cells not exceeding400individuals |
ROX |
0 μl(no ROX),0.2 μl(High ROX) or0.02 μl(Low ROX) |
Ultra-pure water (nuclease-free) |
Add to20 μl |
6.RT-qPCRAmplification conditions:
RT-qPCRAmplification is divided into reverse transcription,Taqenzyme activation, andPCRamplification—three stages. It is recommended to performPCRmelting curve analysis after amplification, especially during the optimization of experimental conditions, in order to distinguish between specific and non-specific amplification as well as primer dimers. After reverse transcription,activateTaqenzyme strictly according to95℃5minuteswill be carried out. The following examplePCRamplification conditions are a two-step method; specific conditions need to be optimized based on the template and primers, particularly the annealing/extension conditions, or amplification can be performed using a3step method.
Reverse transcription: 50℃, 5minutes
Taqenzyme activation:95℃5minutes (a mandatory condition—do not change)
PCRamplification: 40cycles (95℃10 sec,60℃20sec/Signal collection)
Melting curve
Guidelines
1. It is not recommended to alter the amounts of reaction reagents without rigorous validation.
Picture
Picture
Bioactivity
