cDNA synthesis .

Our primaREVERSE BASIC Reverse Transcription Kit contains everything you need for cDNA synthesis from purified total RNA.

The proprietary reverse transcriptase with reduced RNase-H activity and increased thermostability guarantees high specificity and yield of cDNA synthesis. The included Oligo(dT) and Random Hexamer primers allow you to very easily optimize the protocol according to your needs.

Hard Facts .

Ready-to-use complete kit incl. Primer and DNase
Suitable for templates up to 17 kb
High yield due to reduced RNase-H activity
Complete reverse transcription in under 10 minutes
For use with specific primers, random hexamers or oligo(dT) primers.
For complex transcripts the RT can be raised up to 65°C.

Applications .

cDNA synthesis

Fast and easy cDNA synthesis with only one kit. The primaREVERSE RT kit offers high sensitivity and specificity. In addition, it is very flexible and provides high yields with oligo(dT), random hexamers or gene-specific primers.

Gene expression analysis

Gene expression levels in tissues or different cell types can be determined by detecting specific mRNA. For this purpose, the total RNA is first transcribed into cDNA. In the subsequent qPCR with gene-specific primers the respective expression level is determined.

cDNA Sequencing (Diagnostics)

In RNA virus diagnostics a cDNA library is created prior to sequencing. In addition to pathogen detection itself, it can also be used to generate mutation profiles of pathogens.

RNA-seq

As part of an RNA-seq workflow, mRNA enrichment can be performed using reverse transcription in conjunction with oligo(dT) primers. This allows both mRNA selection and RT to be performed in one step, which is particularly useful for small amounts of RNA. It should be noted that a 3′ bias may occur during subsequent sequencing. The result would be an increased number of reads for the 3′ end of the RNAs.

The features .

Simple workflow for maximum cDNA yield .

With the primaREVERSE RT kit, you can transcribe RNA into cDNA in less than 15 minutes.

You do not need to pipette on ice, as the product includes an RNase inhibitor that prevents degradation of total RNA.

Content .

Content of the kit:

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Component	Concentration

10x DNase Incubation Buffer	10x
DNase Enzyme Mix	5 U/µl
Reverse Transcriptase	200 U/µl
5x Reaction Buffer	5x
dNTP Mix	10 mM
Oligo-(dT)20 Primer	100 µM
Random Hexamer Primer	100 µM
RNase Inhibitor	40 U/µl
RNase-free water	–

Kit Packaging:

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Item number	Purchase unit
SL-9540-100	100 reactions (20 µl)
SL-9540-1000	1000 reactions (20 µl)

Sensitive to minute amounts of RNA .

The primaREVERSE Reverse Transcription Kit enables cDNA synthesis even from samples with the smallest amounts of RNA.

From RNA to cDNA in less than 15 minutes .

The primaREVERSE RT Kit allows transcription from RNA to cDNA in less than 15 minutes – with full performance.

With our proprietary ultra-fast reverse transcriptase, cDNA synthesis can even be shortened to as little as 1 minute, with only a minimal reduction in performance.

For complex transcripts, reverse transcription can be extended up to 60 minutes.

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Primer included .

The primaREVERSE RT kit contains Random Hexamer and OligoDT(20) primers so you can make the best choice for your assay.

In many cases, a 1:1 mixture of random hexamer and oligoDT(20) primer or random hexamer primers alone will give you the best results. However, in some assays, it may also be useful to use oligoDT(20) primers that bind to the poly-A tail of the mRNA.

The primaREVERSE RT kit is also ideally suited for the use of gene-specific primers. Details can be found in the manual.

Which primer should I use ?

For most assays, we recommend using either a 1:1 mixture of random hexamer and OligoDT(20) primer or random hexamer primer alone.

gDNA Removal for more accurate target identification .

The primaREVERSE RT kit contains a DNase enzyme mix for removal of genomic DNA.

Removal of genomic DNA from total RNA is generally recommended because even primers that do not bind on genomic DNA can generate a signal in qPCR, see the following figure.

RT Kit Verkaufsunterlagen DNaseTreatment Curve

Figure 1:

qPCR curve plot for GAPDH with RNA-specific primers and an input amount of 1000 ng total RNA. Without DNase digestion, the genomic DNA still present in the sample leads to an amplification signal.
DNase digestion can be used to prevent unwanted amplification.

RT Kit Verkaufsunterlagen DNaseTreatment Cq

Figure 2:

qPCR results (Cq values) with RNA-specific primers and an input amount of 10 ng total RNA. Reverse transcription was performed using OligoDT or random hexamer primers.

Without DNase digestion, the qPCR signal will be distorted. Gene expression appears higher than it actually is.

Made in Germany .

We develop and produce our enzymes and master mixes exclusively ourselves in Germany. You benefit from this in several ways:

Reverse Transcription Kit .

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Downloads .

Manuals

Flyer

Standard protocol

Advanced protocol

Manuals

Flyer

Standard protocol

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Component	Stock concentration	20 µl reaction	Final concentration
RNA Template	–	10 μl	Total RNA: 10 pg up to 5 μg per reaction
			Purified mRNA: 10 pg up to 500 ng per reaction
Primer**	100 µM	1 µl	5 µM ; gene-specific primer: 500 nM *
5x reaction buffer	5x	4 µl	1x
dNTP mix	10 mM	1 µl	500 µM
RNAse inhibitor (optional)	40 U/µl	0.5 µl	1 U/µl
Reverse transcriptase	200 U/µl	1 µl	10 U/µl
RNAse-free water	–	up to 20 µl	–

Incubation at 50-55°C for 1-10 min.
Optional: Heat inactivation at 95°C for 2 min.

* Total RNA: 10 pg – 5 μg; purified mRNA: 10 pg – 500 ng; Sample from DNase Digestion Step
** either Oligo(dT)20, Random Hexamer or 1:1 mix of Oligo(dT)20 and Random Hexamer Primer
*** For gene-specific primers we recommend to use 500 nM final primer concentration

Advanced protocol

Primer/RNA Mix

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Component	Stock concentration	11 µl primer/RNA mix	Final concentration
RNA template or sample from gDNA removal step*	–	up to 10 µl	Total RNA: 10 pg up to 5 μg per reaction
			Purified mRNA: 10 pg up to 500 ng per reaction
Primer**	100 µM	1 µl	5 µM; gene-specific primer: 500 nM*
RNAse-free water	–	up to 11 µl	–
Primer annealing: Incubation at 70°C for 5 min.

* Total RNA: 10 pg – 5 μg; purified mRNA: 10 pg – 500 ng; Sample from DNase Digestion Step
** either Oligo(dT)20, Random Hexamer or 1:1 mix of Oligo(dT)20 and Random Hexamer Primer
*** For Gene-Specific Primers we recommend to use 500 nM final primer concentration

Final Reaction Mix

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Component	Stock concentration	9 µl reaction mix	Final concentration in reaktion (20 µl)
5x reaction buffer	5x	4 µl	1x
dNTP mix	10 mM	1 µl	500 µM
RNase inhibitor (optional)	40 U/µl	0.5 µl	1 U/µl
Reverse transcriptase	200 U/µl	1 µl	10 U/µl
RNase-free water	–	auf 9 µl	–
Mix 9 µl of the reaction mix with 11 µl of primer/RNA Mix after incubation.
Incubation at 50-55°C for 1-10 min.
Optional: Heat inactivation at 95°C for 2 min.

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