Description

Foregene One-Step RT-PCR EasyTM 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, which simplifies the experimental steps, optimizes the experimental program, and improves the experiment efficiency.

RT-PCR EasyTM I(One Step) is a one-step RT-PCR kit specially developed by Foregene, which realizes continuous operation from RT to PCR in the same tube. The operation is simple and fast, which can effectively reduce the pollution during the experiment. 2×RT-PCR EasyTM Mix has strong tolerance, thermal stability, high efficiency and specificity for amplification.

Features

The one-step kit enables reverse transcription and PCR to be carried out in the same tube. It only needs to add template RNA, specific PCR primers and RNase-Free ddH2O.
Real-time quantitative analysis of RNA can be carried out quickly and accurately.
The kit uses a unique Foregene reverse transcription reagent and Foregene HotStar Taq DNA Polymerase combined with a unique reaction system to effectively improve the amplification efficiency and specificity of the reaction.
The optimized reaction system makes the reaction have higher detection sensitivity, stronger thermal stability, and better tolerance.

Kit Contents

Kit components	RT-02011	RT-02012
Kit components	100T (20μl system)	500T (20μl system)
2× RT-PCR EasyTM Mix	1ml	1.7ml×3
RNase-Free ddH2O	1.7ml	1.7ml×3
Instruction Manual	1piece	1piece

Kit component information

2× RT-PCR EasyTM Mix:Foregene Reverse Transcriptase, RNase Inhibitor, Foregene HotStar Taq DNA Polymerase,dNTPs, Mg2+,reaction buffer, optimizer and stabilizer, etc.

Precautions: (Please read the precautions carefully before using the kit)

For templates, it is recommended to use RNA extracted from fresh samples or stored at -80℃ (RNA should avoid repeated freezing and thawing).

In order to avoid RNase contamination, the experiment operation should be carried out in the RNase-Free space; the pipette tips and PCR centrifuge tubes used must be RNase-Free; and disposable gloves and masks should be worn. Before use, put the 2×RT-PCR EasyTM Mix 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 the specificity of PCR amplification.

Transport and storage conditions

1.Transportation conditions

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

2. Storage conditions

RT EasyTM I is stored at -20°C. Store the product in a constant temperature refrigerator at -20°C immediately after receipt. If the storage conditions are appropriate, the product will not degrade any performance during the 1-year validity period.

Template RNA concentration

RT-PCR EasyTM I(One Step):(0.1pg-1μg total RNA)/20μl system

Operation guide

A: Preparation of materials and reagents

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

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

2. Place 2× RT-PCR EasyTM Mix and RNase-Free ddH2O on an ice bath to let it melt naturally, and flick the tube wall to mix well for later use.

B: RT-PCR system preparation

2×RT-PCR EasyTM Mix is convenient and quick to use, avoiding 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 20μl, take 10μl 2×RT-PCR EasyTM Mix solution), add appropriate amount of RNA template and specific primers, and add RNase-Free ddH2O to make up the volume. Refer to Table 1 below for the specific RT-PCR reaction system preparation.

Form 1:RT-PCR system preparation

RT-PCR system additions	Amount	Final Concentration
2× RT-PCR EasyTM Mix	10μl	1×
Forward Primer (10μM)	0.5μl	0.2-0.25μM 1*
Reverse Primer (10μM)	0.5μl	0.2-0.25μM 1*
Template(RNA)	Xμl	0.1pg-1μg
RNase-FreeddH2O	(9-X)μl
Total Volume	20μl

1*: Usually the final concentration of primer is 0.2-0.25μM to get better results. When the reaction performance is poor, the primer concentration can be adjusted within the range of 0.1-0.5μM.

Note: Forward Primer and Reverse Primer are the specific primers for the target gene; 50μl system, please refer to the 20μl system to adjust the reagent dosage proportionally.

C: RT-PCR reaction program setting

After preparing the RT-PCR system with reference to the above table, mix gently (you can use pipette tip to blow gently; 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).

2. Refer to the RT-PCR reaction program settings (Table 2) to set the temperature and time of the reaction.

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

Form 2:RT-PCR reaction system setting

Step	Temperature	Time	Cycles	Content
1	42℃	10-30min	1	RT
2	94℃	5min	1	Predenaturation
3	94℃	10sec	30-40	Denaturation
4	55-65℃	20sec		Annealing
5	72℃	Xmin (2kb/min)		Extension
6	72℃	5min	1	Final extension

Note: The above procedure is for reference only. The actual reaction conditions vary depending on the structural conditions of the template, primers, etc. For RNA templates with complex secondary structures, the reaction temperature for the first step of reverse transcription is recommended to be 50°C. In the specific operation, it is necessary to design the optimal reaction conditions, including annealing temperature, extension time, etc. according to the specific conditions of the target fragment size, the base sequence of the amplified fragment, and the GC content and length of the primer.

Work Flow