Problems with secondary structures of RNA

RNA exists, in contrast to DNA, normally as a single strand molecule, but it may come to formations of secondary structures by refolding of the single strand.
The secondary structures of nucleic acids (DNA and RNA) refer to a 2D-folding of the single strand. Since there is no complementary strand in RNA, the base pairing is done by refolding of the single strand so that complementary segments can mate. The secondary structure is, similar to the double-stranded DNA, formed by hydrogen bonding of the complementary bases. The more GC bases are paired the more stable the secondary structure is. To the secondary structures of RNA belong for example hairpin, stemloop and loop structures (

For an optimal reverse transcription secondary and tertiary structures must be broken up. This requires, depending on the length of the complementary structure and the base pairing (high GC content), increased temperatures. In contrast to HotScriptase RT (M3056) from Genaxxon conventional reverse transcriptases - as M-MuLV (M3042) or the AMV reverse transcriptase – are not thermostable and work best at temperatures around 37°C (up to 42°C). At this low  temperatures many secondary structures of the RNA can´t be broken up. For this, for example, binding sites of the primers at the RNA can become inaccessible (prevention of the whole reverse transcription), or an initiated reverse transcription terminates in a loop structure and can therefore not be completed (incomplete reverse transcription).

With the new HotScriptase RT Polymerase from Genaxxon such problems belong to the past. Reverse transcription and DNA amplification work simultaneously while running a standard PCR protocol. Since the PCR is executed at higher temperatures and at the same time a denaturation step at 95°C is performed, secondary structures or high GC contents are no longer a problem. All steps can be carried out at temperatures above 55-75°C, respective should even carried out at temperatures >57°C.

And it can be even easier using our HotScriptase RT master mix. With our master mix you have to pipette only primers and mRNA to the RT-PCR reaction and start the PCR program. Or if you want to start from cells without laborious isolation and purification of RNA just use our HotScriptase RT Cell master mix. It is proven to be a very neat way to get results from even difficult targets as Flavi viruses (Zika virus, yellow fever virus, dengue virus).

Extract from our protocol for HotScriptase RT Polymerase:

  1. Thaw HotScriptase RT cell master mix at RT or on ice.
  2. Prepare RT-PCR reaction mix according to Table 1.
    The master mix typically contains all of the components needed for RT-PCR except primers and template.
  3. Program the thermal cycler according to the manufacturer’s instructions.
    A typical RT-PCR cycling program is outlined in Table 2. For maximum yield and specificity, temperatures and cycling times should be optimized for each new target or primer pair.
  4. Place PCR tubes in the thermal cycler and start program

Table 1: Recommendations for PCR / Reaction Setup (25µL PCR reaction)



Final concentration

Primer forward (10µM)

Primer reverse (10µM)

HotScriptase RT 2X
Cell master mix

Template/Sample extract*

Nuclease-free water




2µL - 10.5µL

up to 25μL total reaction volume

0.5µM (0.05-1µM)

0.5µM (0.05-1µM)


100 - <10000 cells


 *Recommended final template concentration is between 5ng/µL to 500ng/µL.

Table 2:
Typical “Zero-Step” RT-PCR protocol (an isothermal reverse transcription step is not needed)





Initial denaturation






>57 – 75°C


2 min.

15 sec.

min. 45 sec. (25 – 40 cycles)


 NOTE:      HotScriptase RT does not have an activity optimum at 68°C. For this reason you can use much higher temperatures for elongation if needed (if the primer sequence does allow higher temperatures)

NOTE:      A “Two-Step” as well as “Three-Step” PCR protocol can be used. If primer needs annealing temperatures below 65°C a three step protocol is recommended.

NOTE:      A new RT-PCR is ideally established by running a temperature gradient in order to find the best annealing/extension temperature for each new primer pair.

NOTE:      Optimal PCR protocol times and temperatures may vary depending on the used cycler, the nature of template and the amplicon length.


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