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The synthesis of complementary DNA (cDNA) is fundamental to the study of RNA expression in cells and tissues. RNA is fragile and not compatible with the polymerase chain reaction (PCR). Therefore, an intermediary step is needed to convert RNA to cDNA for analysis by quantitative PCR (qPCR). This qPCR technique utilizes specific primers and polymerase enzyme to amplify targeted sequences of cDNA. Since the cDNA is complementary to the initial mRNA isolate, this technique provides a quantitative readout of mRNA levels in the sample. The need for cDNA is so great that many companies sell cDNA synthesis kits to simplify the procedure.

How cDNA synthesis works

The first step of cDNA synthesis is to denature mRNA at about 70ºC. This is important because it relaxes the secondary structure to allow the reverse transcriptase (RT) enzyme to travel the length of the strand. The next step is a quick drop in temperature to allow an initial primer sequence to bind. Then, the RT attaches and moves from the 3′ to the 5′ end of the RNA strand. It reads each base pair and inserts to corresponding match to extend the primer resulting in cDNA. The RT works optimally at 37ºC, but some newer proprietary RT enzymes can work at temperatures as high as 50ºC. The final step of the reaction is tto heat it up to 70ºC in order to deactivate the RT. An additional RnaseH step can be added to degrade the RNA strands and leave only the cDNA strands in the sample.

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A cDNA synthesis kit contains 4 main ingredients

The cDNA synthesis kits may vary slightly between manufacturers, but the essential ingredients are always included. The major differences lie in the buffer composition and the choice of reverse transcriptase. Manufacturers of cDNA synthesis kits gain their edge by developing proprietary RT enzymes to speed up the process of to increase transcription efficiency. As a result, there are many RT varieties available.

4 main ingredients of a cDNA synthesis kit:

  1. Reverse transcriptase (RT)
  • A traditional RT is M-MuLV, which works at 37ºC. The downfall of this RT is that it becomes deactivated at temperatures above 37ºC. Sometimes higher temperatures are needed to further relax the secondary structure of G-C rich mRNA. To solve this problem proprietary RT enzymes such as superscript III have been developed that can withstand heat up to 50ºC.
  • The length of time the RT stays attached to the RNA template can determine the total time necessary for the reaction. If the RT stays attached longer then it can transcribe the entire cDNA chain at once rather than doing it piece by piece. Newer superscript RT enzymes are available to accomplish this.

 

2. Primer

  • Oligodt primers bind to the 3′ polyA tail.
  • Random primers bind all along the RNA transcript. This is necessary for mRNA that is missing the polyA tail

 

3. dNTPs

  •  These individual nucleotides are used by the RT to extend the cDNA strand.

 

4. Buffer

  • DTT: a reducing agent that breaks disulfide bonds to help break down the secondary structure of RNA
  • MgCl2: a cofactor for the RT.

And the optional ingredients:

5. RNAseOUT to protect RNA from degradation throughout the reverse transcription process
6. RnaseH to clean up the cDNA sample at the end of synthesis by digesting the RNA strands

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Lluis M. Martínez | SEPMAG Chief Scientific Officer

Founder of SEPMAG, Lluis holds a PhD in Magnetic Materials by the UAB. He has conducted research at German and Spanish academic institutions. Having worked in companies in Ireland, USA and Spain, he has more than 20 years of experience applying magnetic materials and sensors to industrial products and processes. He has filed several international patents on the field and co-authored more than 20 scientific papers, most of them on the subject of magnetic particle movement.

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