RNA analysis tools

Engineered M-MLV Reverse Transcriptase Basic Kit, 50 rxns

Engineered M-MLV Reverse Transcriptase Basic Kit includes a rationally designed M-MLV RT that exhibits increased thermal stability and reduced RNase H activity (RNase H) than the wild type M-MLV RT.

(SKU: RN012S)


For detailed product information please refer to the IFU

Product Details

Engineered M-MLV Reverse Transcriptase Basic Kit includes a rationally designed M-MLV RT that exhibits increased thermal stability and reduced RNase H activity (RNase H) than the wild type M-MLV RT.

  • Engineered M-MLV RT remains active up to 50oC increasing the length and yield of cDNA compared to WT version.
  • Able to efficiently detect and produce cDNA in reactions that have less than 100 pg of RNA template.
  • Able to amplify regions up to 8kb.


Gene coding the respective variant is heterologously expressed in E.coli.

Unit Definition:

One unit is defined as the amount of enzyme needed to catalyze the incorporation of 1 nmol dTTP into acid-insoluble material in 10 min under 37°C with poly(rA) and oligo(dT).

Reagents supplied:

RN012S kit
Cat No Description Tubes No.
RN001S Engineered MMLV Reverse transcriptase, 200u/μl, 0.05ml (200 units/μl in 20 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.1 mM EDTA, 50 % glycerol, 0.01% IGEPAL, 1mM DTT) 1
BR055-1 5x Reverse Transcriptase buffer, 1ml (250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2) 1
BR056-0.5 100mM DTT, 0.5ml 1
BR028-0.15 dNTPs mix 10mM each, 0.15ml 1
BR033-1 Nuclease Free Water, 1 ml 1

Reaction guidelines and tips:

Quantities per Reaction (20μL Final Reaction Volume): 

Preparation of Mix A  1 ng -2 μg of total RNA* 
2 μL oligod(T) 18 from 50 μΜ stock  §, §§
1 μl dNTPs mix from 10 mM each stock 
X μl of Nuclease Free Water to 10 μl 
Incubate Mix A at 65ο C for 5 min, then transfer for 1 min on ice (meanwhile prepare Mix B)
Preparation of Mix B 2,5 μl of Nuclease Free Water  
4 μl from 5x RT Buffer 
2 μl DTT from 100 mM stock 
0,5 μl of RNase inhibitor** 
1 μL of Reverse Transcriptase enzyme from stock of 200 U/μl 
Following Step 2, add 10 μL of Mix B to the reaction 
Incubate at 37ο C to 55ο C for 15 min ***  
Heat inactivate the Reverse Transcriptase at 65ο C  for 20 min
Cool down the cDNA to room temperature and use 0,1 – 2 μl in PCR reaction 

* ≤ 0,1 ng may suffice for detecting certain RNA populations that are abundant like e.g. Rubisco transcript in plants. Use 10 times less RNA quantity for coding RNAs enriched substrates. 

** The final concentration of the RNase Inhibitor is 1 U/μL. Since RT enzyme and reagents have been checked for the presence of RNases and DNases, this step may be omitted if the sample is of high RNA quality. 

*** Routine temp for a standard reaction is 42ο C. For most RNA cases tested complete cDNA production was performed within 10-15 minutes independently of the incubation temperature. Optimize depending on your target and provide us with your feedback to get special offers and rewards! 

§ In case of random hexamers, add 2 μl from 50 μM stock per 20 μl RT reactions. Following “step2” incubate for 5 minutes at 25ο C or room temperature before incubating the reaction to 42ο C. 

§§ In case of gene specific primer, add 2 μl from 2 μΜ primer stock. Incubate at 50ο C for cDNA production. Optimize to your case depending on primer’s Tm. Incorporate 5-10 degrees below the Tm of your oligo. Do not incubate at temperatures higher than 55ο C.  

Functional Quality Control:

Two step cDNA synthesis reaction in order to amplify an 831-bp region using 1ng Hela RNA as substrate. The resulting PCR product is visualized as a single band on a midori-stained agarose gel.

Other Quality Controls

Tested extensively for the absence of DNases and RNases and E.coli DNA contamination.


Shipped on blue ice or dry ice

Storage conditions

Store at -20οC ± 5οC

Shelf life:

12 months from production

RN001S can efficiently reverse-transcribe in 15 minutes!

1 μg of HeLa RNA was reverse transcribed for either 60, 30 or 15 minutes followed by immediate heat inactivation of the RT enzyme.

One standard reaction of 60 minutes was set in parallel with two different reactions (A and B) for each time point.

1 μl of each resultant cDNA was subjected to qPCR analysis using ΕQ 2x qPCR Μaster Μix Green kit, w/o ROX (TM) (RN014S)

Primers specifically designed for qPCR for one low and one high copy gene were included in the mixes.

PCR target site for the low-copy gene is located near the 5’ end, targeting the area 234-364 of a 12776 bp long mRNA, suggesting the full-length cDNA production

nTPM : normalized Transcripts per Million

i. Low copy gene*: 7.8 nTPM in HeLa cells (based on Human Atlas website)

ii. High copy gene*: 5924.3 nTPM in HeLa cells (based on Human Atlas website)

* Expression levels’ annotation (low and high) has been performed according to the Expression Atlas website (EMBL-EBI)

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