QuantiNova Reverse Transcription Kit

ユニークなQuantiNova Reverse Transcription Kit はgDNA除去及び付属のインターナルコントロールを用いることで、迅速かつ簡便なcDNA合成のプロトコールを提供します。RNAサンプル中に混入しているゲノムDNAはgDNA Removal Mixによって除去され、かつ付属のInternal Control RNAによって、確実な逆転写と増幅を検証するために使用できます。QuantiNova Reverse Transcription Kit は迅速かつ効果的なcDNA合成を提供します。合成されたcDNAはリアルタイムPCRでの使用に至適化され、mRNA転写物の全ての領域を確実に定量できます。

Contaminating genomic DNA in RNA samples is effectively and rapidly removed with the unique gDNA Removal Mix. Elimination of genomic DNA is crucial for accurate gene expression results and when design of RNA-specific primers or probes is not always possible, for example, when analyzing single-exon genes. With gDNA Removal Buffer, time is saved and costs are reduced, since a separate DNase digestion during or after purification of RNA samples is not required.

The newly developed internal control is a defined transcript (RNA molecule) that can simply be added to your samples and transcribed into cDNA. It is intended to report instrument or chemistry failures, errors in assay setup and the presence of inhibitors. The expected range of C_q (C_T) values can be used to test successful reverse transcription/amplification and the reproducibility of your results.

The high RNA affinity of the QuantiNova Reverse Transcriptase enables high yields of cDNA from any RNA template. Difficult templates, such as those with high GC-content or complex secondary structure, are also successfully reverse transcribed. The Reverse Transcription Mix contains a specially optimized mix of oligo-dT and random primers that enable cDNA synthesis from all regions of RNA transcripts, including 5' regions. The kit provides high yields of cDNA template for real-time PCR analysis, regardless of where the amplified target region is located on the transcript, and provides greater sensitivity in the detection of low-abundance genes.

The QuantiNova Reverse Transcription Kit offers accurate results over a wide range of input amounts, including up to 5 µg RNA in the standard protocol, which can be doubled to accommodate exceptionally large amounts of RNA.

The QuantiNova Reverse Transcriptase has a high affinity for RNA and is capable of cDNA synthesis from a wide range of RNA amounts (10 pg – 5 μg) (see figure “ Precise quantification from 5 µg – 10 pg RNA). The kit provides high yields of cDNA template for real-time PCR analysis, regardless of where the amplified target region is located on the transcript. Even difficult templates, such as those with high GC-content or complex secondary structure, are successfully reverse transcribed. The QuantiNova RT Buffer has also been optimized to be compatible with real-time PCR buffers. 

To obtain accurate results in real-time RT-PCR gene expression assays, it is important that only cDNA is amplified and detected. Interference by genomic DNA can effectively and rapidly be removed using the gDNA Removal Mix (see figure “ Efficient removal of contaminating gDNA ensures precise quantification of transcripts). Time is saved and costs are reduced, since a separate DNase digestion during or after purification of RNA samples is not required. Also, designing RNA-specific primers or probes is not necessary. 

Detecting variations in cDNA synthesis allows you to check the reproducibility of your results. The newly developed internal control is a defined transcript (RNA molecule) that can be optionally added to your samples and transcribed into cDNA. It is intended to report instrument or chemistry failures, errors in assay setup and the presence of inhibitors. Because the internal control behaves comparable to real transcripts, it can be used to confirm successful reverse transcription and amplification in subsequent qPCR (see figure “ Internal control reliably detects presence of inhibitors).

Genomic DNA removal and cDNA synthesis take only 20 minutes with the QuantiNova Reverse Transcription Kit. The kit includes everything you need for fast cDNA synthesis. 

Purified RNA is briefly incubated in gDNA Removal Mix to effectively remove contaminating genomic DNA. In contrast to other methods, the RNA sample is then used directly for reverse transcription in combination with a master mix prepared from the Reverse Transcription Mix plus Reverse Transcription Enzyme. With the QuantiNova Reverse Transcriptase, RNA can be transcribed at low temperatures, even through complex 2° degree structure. This ensures that the RNA will stay intact as the reaction takes place at 42°C and is then inactivated at 85°C. Additional steps for RNA denaturation or RNase H digestion are not necessary.

The QuantiNova Reverse Transcription Kit allows highly efficient and sensitive real-time RT-PCR for all types of starting materials, including laser-microdissected samples and tissue biopsies.