To create far more benefit for customers is our company philosophy; customer growing is our working chase for Super Lowest Price China Same Quality as Qiagen Diagnostic Rt-PCR Testing Reagent Kit for  (ORF1ab) /Influenza a&B with CE, We welcome you to definitely check out our manufacturing unit and look ahead to creating welcoming business relationships with consumers at your home and abroad within the in the vicinity of long run.
To create far more benefit for customers is our company philosophy; customer growing is our working chase for China Qiagen, Multiplex Reagent, During in 11 years, We have now participated in more than 20 exhibitions, obtains the highest praise from each customer. Our company has been devoting that “customer first” and committed to helping customers expand their business, so that they become the Big Boss !

Descriptions

This kit uses a unique lysis buffer system that can quickly release RNA from cultured cell samples for RT-qPCR reactions, thereby eliminating the time-consuming and laborious RNA purification process. The RNA template can be obtained in just 7 minutes. The 5×Direct RT Mix and 2×Direct qPCR Mix-SYBR reagents provided by the kit can quickly and effectively obtain real-time quantitative PCR results.

5×Direct RT Mix and 2×Direct qPCR Mix-SYBR have strong inhibitor tolerance, and the lysate of the samples can be used as the template for RT-qPCR directly. This kit contains the unique RNA high-affinity Foregene reverse transcriptase, and Hot D-Taq DNA polymerase, dNTPs, MgCl₂, reaction buffer, PCR optimizer and stabilizer.

Specifications

200×20μl Rxns, 1000×20μl Rxns

Kit components

Features&advantages

■  Simple and effective : with Cell Direct RT technology, RNA samples can be obtained in just 7 minutes.

■ The sample demand is small, as low as 10 cells can be tested.

■ High throughput: it can quickly detect RNA in cells cultured in 384, 96, 24, 12, 6-well plates.

■ DNA Eraser can quickly remove released genomes, greatly reduce the impact on subsequent experimental results.

■ Optimized RT and qPCR system makes the two-step RT-PCR reverse transcription more efficient and PCR more specific, and more resistant to RT-qPCR reaction inhibitors.

Kit application

Scope of application: cultured cells.

- RNA released by sample lysis: only applicable to the RT-qPCR template of this kit.

- The kit can be used for the following purposes: gene expression analysis, verification of siRNA-mediated gene silencing effect, drug screening, etc.

Diagram

Storage and Shelf life

Part I of this kit should be stored at 4℃; Part II should be stored at -20℃.

Foregene Protease Plus II should be stored at 4℃,do not freeze at -20℃.

Reagent 2×Direct qPCR Mix-SYBR should be stored at -20℃ in the dark; if used frequently, it can also be stored at 4℃ for short-term storage (use up within 10 days).To create far more benefit for customers is our company philosophy; customer growing is our working chase for Super Lowest Price China Same Quality as Qiagen Diagnostic Rt-PCR Testing Reagent Kit for  (ORF1ab) /Influenza a&B with CE, We welcome you to definitely check out our manufacturing unit and look ahead to creating welcoming business relationships with consumers at your home and abroad within the in the vicinity of long run.
Super Lowest Price China Qiagen, Multiplex Reagent, During in 11 years, We have now participated in more than 20 exhibitions, obtains the highest praise from each customer. Our company has been devoting that “customer first” and committed to helping customers expand their business, so that they become the Big Boss !

QuickEasyTM Cell Direct RT-qPCR Kit -Taqman

Cat.No.DRT-01021/01022

For cell direct RT-qPCR using ≤ 1000,000 cells

Product introduction

This product uses a unique lysis buffer system to quickly release RNA from cultured cell samples for RT-qPCR reactions, eliminating the time-consuming and laborious RNA purification process, and only 7 minutes to obtain the required RNA template, with the 5× Direct RT Mix, 2× Direct qPCR Mix-Taqman provided by the kit can quickly and efficiently obtain real-time quantitative PCR results.

5× Direct RT Mix and 2× Direct qPCR Mix-Taqman have strong inhibitor tolerance and can perform efficient reversal and specific amplification using the lysate of the sample to be measured as a template. The reagent contains Foregene Reverse Transcriptase, Hot D-Taq DNA Polymerase, dNTPs, MgCl₂, Reaction Buffer, PCR Optimizer and Stabilizer, which can be used with lysis buffer to quickly and easily detect samples, and has the characteristics of high sensitivity, specificity and stability.

Product features

Simple, effective Cell Direct RT technology that takes as little as 7 minutes to get RNA samples.

Sample requirements are small, and a minimum of 10 cultured cells can be used for experimentation.

High throughput for rapid RNA acquisition of cultured cells such as 384, 96, 24, 12, and 6-well plates.

DNA Eraser is able to quickly remove released genomes, greatly reducing the impact on subsequent experimental results.

Optimized RT and qPCR systems enable two-step RT-PCR with more efficient reverse transcription, specificity, and stronger RT-qPCR reaction inhibitor tolerance.

Kit application

Scope of application: Cultured cells.

Sample lysis interpreted RNA: used only as a two-step RT-qPCR template.

Kits can be used for the following purposes: gene regulatory expression analysis, allele testing, drug screening, etc.

Kit limitations

Amplified fragments ≤ 300 bp.

Kits are used to freshly culture cells.

Product quality control

According to the FOREGENE’s Total Quality Management System, each batch of Cell Direct RT-qPCR series kits is rigorously tested multiple times to ensure the reliability and stability of the quality of each batch of kits.

Kit contents

*:Cell Lysis, 5×Direct RT Mix, 2× Direct qPCR Mix-Taqman can be purchased separately,details are provided in Appendix 1 (PAGE 13).

Storage conditions

1. Shipping Conditions

The whole process of low temperature ice pack box transportation, to ensure that the kit is in the <4 °C state.

2. Storage conditions

Store part I at 4°C and Part II at -20°C.

The Foregene Protease Plus II should be stored at 4°C,not frozen at -20°C.

Reagent 2× Direct qPCR Mix-Taqman is stored at -20°C, or at 4°C for short-term use if used frequently (within 10 days).

Kit component information

Buffer CL: Provides the environment required for cell lysis reactions.

Buffer ST: Terminates the active substance in the lysate to avoid effects on subsequent RT.

DNA Eraser: DNA remover, the effect of removing the genome on subsequent experiments.

5× Direct RT Mix: Contains high RNA affinity Foregene Reverse Transcriptase, RNase Inhibitor, dNTPs, stabilizers, enhancers, optimizers, and reverse transcription primers for optimal alignment (Random Primer, Oligo(dT) ₁₈ Primer).

Foregene Protease Plus II: In the context of lysis buffer, cells are lysed to release nucleic acids.

2× Direct qPCR Mix-Taqman: This reagent contains Hot D-Taq DNA Polymerase, dNTPs, MgCl₂, reaction buffer, PCR optimizer, and stabilizer.

20× ROX Reference Dye: Generally used on Real Time PCR amplification instruments of ABI, Stratagene and other companies, it is used to adjust the difference between PCR tubes and tubes caused by PCR dosing errors. The 20× ROX Reference Dye concentration required for different instruments is different, and the user can add it according to the recommended concentration of the instrument.

RNase-Free ddH₂O: RNase-free sterilized ultra pure water for two-step RT-qPCR reactions.

Precautions: (Be sure to read the precautions carefully before using the kit)

Pay attention to the operation method of the experiment to avoid cross-contamination between samples.

Pay attention to the cleanliness of the experimental environment and utensils to avoid RNase contamination and RNA degradation.

Take fresh or well-preserved cell samples and never use repeated freeze-thawed cell samples.

5× Direct qPCR Mix,2× Direct qPCR Mix-Taqman should avoid repeated freeze-thaw, otherwise it will affect reverse transcription and PCR efficiency.

Preparations before operation

Be sure to read the instructions carefully before using this kit. The Cell Direct RT-qPCR Kit is simple, convenient, and fast to operate, and the instructions provide complete information about the entire kit and how to use it correctly. Please prepare the necessary experimental materials and equipment before use.

Experimental materials and equipment

◆ Culture cells.

◆ 1.5 ml or 2 ml, RNase-/DNase-Free centrifuge tube, RNase-/DNase-Free tip, 0.2 ml sterile qPCR tube.

◆ qPCR machine, pipette, tabletop centrifuge (≥13,400×g) (depending on experimental needs), etc.

Safety

◆ This product is for scientific research purposes only, please do not use it for pharmaceutical, clinical, food and cosmetic purposes.

◆ When using chemicals, wear appropriate lab clothes, gloves, protective glasses, etc.

Operation guides

Cell Lysis systems, RT systems, and qPCR reaction solution supplement packs can be purchased separately, for details in Appendix 1 (PAGE 13).

Operation guide

A: Sample RNA release

1.Cells were pretreated: Wash the cell culture plate with cold PBS, then lyse the cells (10-10⁶), 10⁶ than the amount of cells, is recommended Foregene the Cell an RNA Isolation Kit the Total (DE-03111) or Animal Total RNA Isolation Kit (DE-03011) for RNA extraction and purification.

1.1. Adherent cells (24- well plate as an example)

1.1.1. Determine the number of cells in each well, determine that the number of cells is 1 × 10⁵, and use a pipette to remove the culture medium from the culture dish.

1.1.2. Add 200 μl of pre-chilled 1 × PBS to each well. Do not pipet repeatedly and remove PBS from the wells. Tilt the plate and remove as much PBS as possible. Proceed to step 2.

1.1.3. Different cell culture dish or a reference number table 1-1 in a cell culture dish was added precooled 1 × PBS for cell washing.

Table1-1: PBS dosage for different numbers of cells

Note：To ensure a firm adherent cells，a large number of cell loss avoided when washing .

1.2. Suspension cells or adherent cells cultured in non-porous plates

1.2.1. Adherent cells cultured in non-multi well plates (suspension cells start from the next step 1.2.2), collect and separate the cells according to the normal cell collection method, and place them in a culture plate or centrifuge tube; if trypsinization is used, require centrifugation to collect the cells and to remove residual trypsin, added PBS resuspended cells into individual cells to disperse the cells.

1.2.2. After the number of cells counted, aliquoted cells 1×10⁵ one to centrifuge tubes, collect cells by centrifugation at 1000 × g for 10 min.

1.2.3. Add 200 μl PBS to the centrifuge tube, do not pipet repeatedly, and directly aspirate the PBS. proceed to step 2. (If difficult to precipitate and the cells were resuspended again, may be performed 1000×g centrifuged 10 min after discarding the supernatant, the cell pellet proceed to step 2)

2.Cell lysis: Remove Buffer CL, its temperature equilibrated to room temperature, DNA Eraser and Foregene Protease Plus II, according to the following table 1-2 prepared lysis system: (Lysis solution is ready for use).

Table1-2: cleavage system preparation (Note: in the preparation on ice)

3.( 24 –well plate as an example ) Pipette 200 μl of cell lysis master mix into each well, Repeatedly blow 5-10 times, Incubate at room temperature (20-25 ℃ ) for 5 min .

Note：To avoid the formation of bubbles, please when pipetting pipette scale was adjusted to 200μl or less. The cells may appear cloudy after lysis, which is normal.

4.(24 –well plate as an example ) is added in the liquid 20 μl Buffer ST ( different lysis systems Buffer ST added in an amount shown in Table 1-3) , repeated pipetting 5-10 times, at room temperature(20-25 ℃ ) were incubated for 2 min .

Note： The pipette tip disposed below the surface, ensuring that the lysate was added，to avoid the formation of bubbles, please when pipetting pipette scale was adjusted to 200μl or less.

Table 1-3：Add Buffer ST

5.The lysate is used for subsequent RT-qPCR experiments. If the subsequent experiments cannot be performed in time, please keep it on ice for no more than 2hr, and store at -20℃ or -80 ℃ ( no more than three months ).

B: RT system preparation

1.Take out 5 × Direct RT Mix and place it on an ice bath, let it melt naturally, and gently mix it for later use; take out RNase-Free ddH2O and melt it and place it on an ice bath for later use. Prepare the reaction system on ice according to Table 2-1 below.

Table 2-1: Preparation of RT reaction system

2.After completion of system formulation, gently mixed and centrifuged briefly in the following       table 2 -2 reaction conditions RT reaction.

Table 2-2: RT Reaction condition setting

3.After completion of the reaction, the reaction product was placed directly on ice for  qPCR , please put the long-term preservation -20℃ or -80 ℃ .

Note: Due to the use of non-purified template, white precipitates may appear in the reverse transcription product. This is a normal phenomenon. Centrifuge the supernatant immediately for subsequent experiments.

The resulting RT reaction solution is added to the next Step Real Time PCR reaction systems, it is recommended to add amounts ranging from 10-30% of the reaction system.

C: qPCR reaction system preparation

1.Appropriate amount of B prepared in step cDNA template according to the following table 3-1 to prepare a reaction system.

Note: The amount of cDNA template accounts for 10-30% of the qPCR system. For example, in a 20μl qPCR system, add 2-6 μl of lysis buffer, but not more than 6 μl.

2.The optimization good qPCR conditions (Annealing temperature，etc.) for qPCR reaction (The reaction conditions given at Table 3-2 ).

Note: Try to use optimized conditions for qPCR reactions to get better results.

Table 3-1: Preparation of PCR reaction system

1*: Primer concentration can be adjusted in the range of 50-900 nM when primer reaction performance is poor.

Note: The qPCR system can be adjusted according to experimental needs and fluorescence cycler model. For qPCR in a 50 μl system, adjust the reagent dosage proportionally according to the 20 μl system.

2*: Select the appropriate final concentration of ROX Reference Dye according to the fluorescence quantitative thermal cycler. The most appropriate ROX Reference Dye concentrations for common fluorescence quantitative cyclers are shown in the table below:

Table 3-2: qPCR reaction conditions are provided

Note: In order to obtain the best qPCR effect, gradient PCR can be used to optimize the reaction conditions for different templates and different primers. PCR reaction conditions vary depending on the fluorescence analyzer, template, primer, etc. In the specific operation, the optimal reaction conditions need to be designed according to the specific conditions of the fluorescence quantitative thermal cycler, template type, size of the fragment of interest, base sequence of the amplified fragment and GC content and length of primers, including annealing temperature, reaction time, etc.

Real Time PCR primer design principles

Forward Primer and Reverse Primer

For Real Time PCR, primer design is very important. Primers are related to the specificity and efficiency of PCR amplification, and can be designed with reference to the following principles:

◆ Primer length: 18-30bp.

◆ GC content: 40-60%.

◆ Tm value: Primer design software, such as Primer 5, can give the Tm value of the primer. The Tm values of the upstream and downstream primers should be as close as possible. The Tm calculation formula can also be used: Tm = 4 °C (G + C) + 2 °C (A + T). When performing PCR, a temperature below the primer Tm value of 5 °C is generally selected as the annealing temperature (the corresponding increase in the annealing temperature can increase the specificity of the PCR reaction).

◆ Primers and PCR products:

◆ Design primer PCR amplification product length is preferably 100-150bp.

◆ Design primers in the secondary structural area of the template should be avoided as much as possible.

◆ Avoid the formation of 2 or more complementary bases between the 3′ ends of upstream and downstream primers.

◆ Primer 3′ terminal base can not be present with 3 additional consecutive G or C.

◆ The primers themselves cannot have complementary structures, otherwise a hairpin structure will be formed, affecting PCR amplification.

◆ ATCG should be distributed as evenly as possible in the primer sequence, and the 3′ terminal base should be avoided as T.

Appendix 1：Cell Direct RT-qPCR Kit component supplement pack

1.Cell Lysis Solution