Product Information

Product Description

      This product uses a new generation of reverse transcriptase with multiple point mutations using molecular evolution technology, which greatly improves thermal stability and reverse transcription efficiency. 5×HRbio™ III RT Master Mix is a one-tube reverse transcription premix containing all the reagents required for reverse transcription (HRbio™ III Reverse Transcriptase, RNase Inhibitor, Random primers, Oligo dT Primer, dNTP Mixture, Buffer). You only need to add template RNA and water to react. It makes cDNA synthesis more convenient and faster, and is particularly suitable for two-step Real Time PCR detection after cDNA synthesis. Usually, experiments such as Real Time RT-PCR require DNase I digestion to remove residual genomic DNA (gDNA) in RNA, but traditional DNase I treatment is complicated and easily causes RNA degradation and loss. This kit uses a special gDNA Remover with DNA decomposition activity. Only one step is required to complete genome removal and reverse transcription reaction at the same time, which greatly simplifies the operation steps and avoids the risk of sample contamination and RNA degradation caused by complex sample addition process.

Product Usage

First-strand cDNA synthesis. Can be used for the detection of high-copy and low-copy genes.

Features

1. The new generation of reverse transcriptase has greatly improved thermal stability and reverse transcription efficiency.

2. The fully premixed reverse transcription mix only requires one step to add gDNA Remover, template RNA and water to achieve cDNA synthesis and genomic DNA removal at the same time. Reverse transcription can be completed quickly and easily in 15 minutes.

3. The volume of RNA template can be added up to 80% of the total volume, which is very suitable for reverse transcription reactions of low-concentration RNA templates.

4. Premixed Mix does not freeze at -20°C, which reduces thawing and mixing time and makes it easier to use.

5. This product has specially optimized oligo dT and N6 random primer ratios for qPCR, so that cDNA synthesis can start from various regions of the RNA transcript and have the same reverse transcription efficiency, which maximizes the authenticity and repeatability of qPCR results.

Product composition

Transportation and storage methods

Transported by dry ice. Store at -20℃, avoid repeated freezing and thawing, valid for 1 year.

Precautions

1) Avoid RNase contamination.

2) To ensure successful reverse transcription, it is recommended to use high-quality RNA samples.

3) 5×HRbio ™ III RT Master Mix and gDNA Remover contain glycerol, which is very viscous. The solution is easily adsorbed to the tube wall and the outside of the pipette tip, resulting in loss. Please centrifuge before use and avoid loss due to adhesion to the outer wall of the pipette tip. The enzymes contained in 5×HRbio™ III RT Master Mix and gDNA Remover are in excess. Even if 5×HRbio™ III RT Master Mix is used at 3.6 μl-3.8 μl and gDNA Remover is used at 0.8 μl-0.9 μl each time, it will not affect the effect.

How to use

 First-strand cDNA synthesis (using 20 μl reaction system as an example, 10 μl reaction system can also be used)

1. Thaw the template RNA, gDNA Remover, and 5×HRbio™ III RT Master Mix on ice; thaw the RNase free H2O at room temperature (15-25°C) and quickly place on ice after thawing. Before use, flick or vortex each solution to mix well. Centrifuge briefly to collect the liquid remaining on the tube wall to the bottom of the tube.

2. Add the following ingredients to the RNAse free tube: (It is recommended to use PCR tubes on ice and place in a PCR instrument for reaction)

* Total RNA should not exceed 2 μg, and mRNA should not exceed 200 ng (20 μl system)

3. Use a pipette to gently mix (total volume 20 μl)

If the mRNA template used is derived from eukaryotic cells (such as human or mouse tissue cells) and contains a Poly(A) tail structure, incubate at 42°C for 15 min.

If the mRNA template used is derived from prokaryotic cells (bacteria) or viruses and does not contain a Poly(A) tail structure, incubate at 25°C for 10 min and then at 42°C for 15 min.

Note: If the template has complex secondary structure or high GC regions, you may try increasing the reaction temperature to 50°C-55°C (prepared at 42°C for 2 minutes to remove genomic DNA) to help increase yield.

Heat at 4.85°C for 5 sec to inactivate HRbio™ III Reverse Transcriptase and gDNA Remover.

5. The obtained cDNA product can be used immediately for qPCR reaction, or stored at -20°C and used within half a year; for long-term storage, it is recommended to store at -70°C after aliquoting. Repeated freezing and thawing of cDNA should be avoided.

RT-qPCR

      Take an appropriate amount of reverse transcribed cDNA product (generally no more than 1/10 of the qPCR reaction volume) as a qPCR template and perform the next step of fluorescent quantitative PCR according to the manufacturer's instructions for fluorescent quantitative PCR reagents. If the expressed gene content is abundant, the cDNA template can be appropriately diluted according to the actual situation.

Precautions

1. Avoid RNase contamination.
2. To ensure successful reverse transcription, it is recommended to use high-quality RNA samples.
3. 5×HRbio™ III RT Master Mix and gDNA Remover contain glycerol, which is very viscous. The solution is easily adsorbed to the tube wall and the outside of the pipette tip, resulting in loss.
Please centrifuge before use and avoid loss due to adhesion to the outer wall of the pipette tip. The enzymes contained in 5×HRbio™
III RT Master Mix and gDNA Remover are in excess. Even if 5×HRbio™ III RT Master Mix is used at 3.6 μl-3.8 μl and gDNA Remover is used at 0.8 μl-0.9 μl each time,
it will not affect the use effect.

4. This product is for scientific research purposes only!