RT-QPCR EasyTM Taqman One Step Real Time RT-PCR Master Mix

RT-qPCR EasyTM (One Step)-Taqman

For real time PCR using cDNA, purified DNA

Product introduction

Foregene One-Step RT-PCR Easy series products realize the integrated reaction from RNA to double-stranded DNA, that is, reverse transcription and PCR amplification are completed in the same reaction centrifuge tube and the same reaction system, thus the experimental steps simplified, the experimental plan optimized, and the experimental efficiency improved.

RT-qPCR EasyTM (One Step)-Taqman using Foregene HotStar Taq DNA Polymerase, the optimized Taqman qPCR system can quickly and specifically perform Real Time RT-qPCR quantitative detection of trace RNA templates.

Transport and storage conditions

• Transportation conditions: The whole process is transported in a low-temperature ice box to ensure that the kit is in a state of <4°C.
• Storage conditions: The kit is stored at -20℃. Store the product in a constant temperature refrigerator at -20℃ immediately after receipt. If the storage conditions are appropriate, the product will not degrade any performance during the 1-year validity period.

Kit component information

• 2× RT-qPCR EasyTM Mix-Taqman: Foregene Reverse Transcriptase, RNase Inhibitor, Foregene HotStar Taq DNA Polymerase, optimized ratio of dNTPs, Mg2+, stabilizer, enhancer, and optimizer.
• ROX Reference Dye: Generally used on Real Time PCR thermal cyclers of companies such as ABI, Stratagene, etc., to adjust the difference among PCR tubes caused by PCR sample loading errors. The concentration of ROX Reference Dye required by different instruments is different, and the user can add it according to the recommended concentration of the instrument.

Precautions:

u Reagents should avoid repeated freezing and thawing, otherwise the performance of the reagents will decrease or become invalid.

u The reagents in the kit should be protected from light and stored at -20°C.

u It is recommended to use fresh sample extraction or template RNA stored at -80°C (RNA should avoid repeated freezing and thawing).

u In order to avoid RNase contamination, please perform the experiment in the RNase-Free space; the pipette tips and PCR tubes used must be RNase-Free; and wear disposable gloves and masks.

u This kit must be used with specific primers for experiments. Please select the specific primers for the gene to be amplified according to the needs of the experiment.

u Before use, put 2× RT-qPCR EasyTM Mix-Taqman on ice to completely melt, flick and mix well before use; the preparation of the system should be operated on an ice bath to improve the performance of the kit and increase PCR amplification.

Template RNA concentration

RT-qPCR EasyTM (One Step)-Taqman:(1 pg-100 ng total RNA)/20 μl system.

Operation guide

A:Preparation of materials and reagents

• Prepare the prepared RNA template (it is recommended to use Foregene Total RNA Isolation Kit series kits to extract and purify RNA), specific primers (10 μM) and related consumables and instruments.

Note: Please ensure the integrity of RNA and try to use RNA extracted from fresh samples.

• Put 2× RT-qPCR EasyTM Mix-Taqman, RNase-Free ddH2O, and 20× ROX Reference Dye (if necessary) on ice to let it melt naturally, and flick the tube wall to mix until use.

B:RT-qPCR system preparation

2× RT-qPCR EasyTM Mix-Taqman is convenient and quick to use, and avoids pollution during operation and experimental errors caused by multiple preparations of the reaction system to the greatest extent. When using, only half the volume of the reaction system (for example, if the reaction system is 20 μl, take 10 μl 2× RT-qPCR EasyTM Mix-Taqman solution), add appropriate amount of RNA template and specific primers, and add RNase-Free ddH2O to make up the volume. The specific RT-qPCR reaction system preparation can refer to Table 1 below.

Table 1: RT-qPCR system preparation

Note: Forward Primer and Reverse Primer are specific primers for the target gene. The qPCR system can be adjusted according to experimental needs and PCR model. For the final concentration of most primers, we recommend 400nM. Please adjust the dosage of the specific primer and Probe according to the prepared concentration according to our recommended final concentration. For qPCR with 50μl system, please refer to the 20μl system to adjust the amount of reagents proportionally.

1*:Choose the appropriate final concentration of ROX Reference Dye according to different quantitative PCR instruments. The optimal concentration of ROX Reference Dye for common quantitative PCR machines is shown in the following table:

C:RT-qPCR reaction system setting

• After preparing the RT-qPCR system with reference to the above table, mix gently (you can use a pipette tip to gently pipette; you can also mix on a vortexer and centrifuge briefly to collect the liquid scattered on the tube wall or tube cap, and place on ice box for later use).
• Refer to the RT-qPCR reaction program settings (Table 2) to set the temperature and time of the reaction.

Note: In order to ensure the activity of 2× RT-qPCR EasyTM Mix-Taqman and improve its amplification efficiency, it is best to prepare the RT-qPCR reaction system after setting up the PCR instrument program, so that the reaction program can be entered immediately after the system preparation is completed.

• In order to get the best qPCR effect, gradient PCR can be used to optimize the reaction conditions for different templates and different primers.

Note: The extension temperature range of Foregene Hotstar Taq DNA Polymerase provided in this kit is: 60-72℃, and the best extension temperature is 72℃.

The following is an example of RT-qPCR reaction conditions. It is recommended to use a two-step method for PCR reaction. When the template concentration is too low to cause non-specific amplification, low primer Tm value leads to low amplification efficiency or poor amplification curve repeatability, etc., it is recommended to try the three-step method for PCR reaction. Refer to Table 2-1 (two-step method) and Table 2-2 (three-step method) for the setting of RT-qPCR reaction conditions.

Table 2-1: RT-qPCR reaction program setting (two-step method)

Table 2-2: RT-qPCR reaction program setting (three-step method)

1*: Reverse transcription time can be adjusted according to experimental needs. General endogenous genes such as β-Actin only need 10 minutes; to detect specific expressed genes, reverse transcription time can be appropriately extended as needed.

2*: Set a specific time according to the length of the amplified target fragment. The amplification speed of Foregene HotStar Taq DNA Polymerase is 2kb/min

Note: PCR reaction conditions vary depending on the structural conditions of the template, primers, etc. For RNA templates with complex secondary structures, it is recommended to use 42℃ for the first step of reverse transcription. In the specific operation, it is necessary to design the optimal reaction conditions according to the specific conditions such as the size of the target fragment, the base sequence of the amplified fragment and the GC content and length of the primer, including annealing temperature, extension time, etc.

Work Flow: