cDNA Synthesis by RT-qPCR:Principle ,Procedure, Components’ Functions

Introduction to cDNA Synthesis      

Complimentary deoxyribonucleic acid (cDNA) is synthesized from RNA using reverse transcription PCR. cDNA is actually complimentary strand of a mature mRNA (only exons, starts with 5’ AUG and ends with poly-A tail). As cDNA is synthesized from mRNA, it does not contain any introns and have DNA bases i.e. GUAC rather than GUAT. cDNA is also single stranded in this case and can be used as template directly for quantitative real time PCR. Do not mix cDNA with C-DNA (a form of DNA which can be observed under certain conditions such as relatively low humidity and the presence of certain ions, such as Li+ or Mg2+). cDNA library is collections of cells in which vectors are present containing cDNA of specific gene incorporated into it. The first strand cDNA synthesis using commercial kits is the most popular method in cell culture labs to study expression level of specific genes. cDNA synthesis is all about setting a PCR reaction with different ingredients from traditional PCR. RT-PCR can either be one-step or two-step. Following is the principle and procedure of two-step RT-qPCR in which the first step includes only the reverse transcription and second step is quantitative real time PCR.

Following are the components of reverse transcription to synthesize cDNA:

1. RNA Template
2. Primers
3. Reaction Buffer
4. RNase Inhibitor
5. dNTPs
6. Reverse Transcriptase Enzyme
7. Nuclease Free Water

All the above constituents are mixed with perfect concentration and volume. Mostly, the final volume of the RT-PCR after adding all the components is 20ul.

 To better understand cDNA synthesis, you should first read RNA isolation by TRIzol reagent.

Principle of cDNA synthesis by Reverse Transcription

First of all, only the template RNA and primers are mixed with nuclease free water in a PCR tube and kept in thermal cycler at approximately at 65°C to remove the secondary structure present in RNA sample. After incubation, PCR tube is quickly placed on ice. This is very crucial step because primers bind to RNA at low temperature (Figure 01). After this, reaction buffer, dNTP mix, RNase inhibitor and suitable reverse transcriptase is added to the mixture. This final mixture is centrifuged for few seconds to mix all the components completely and kept in the incubator for 60min. In this period, the reverse transcriptase adds dNTPs to synthesize cDNA by using RNA as template. Final cDNA is obtained which is complementary to RNA sample, single stranded and obviously has DNA bases.

NOTE: DNA is not amplified in at this step because this was just reverse transcription of our RNA sample.

Functions of Components

RNA Sample

The starting RNA sample can be

Mature mRNA

If you have extracted the mRNA containing 5’ AUG and 3’ poly-A tail, you need to add 500ng of your mRNA sample to start making cDNA by reverse transcription.

Total RNA

The amount of total RNA which is used for cDNA synthesis is 0.1ng to 5ug. But mostly 1000ng (1ug) is used for reverse transcription PCR. To select the amount of RNA can be tricky because you have specific concentration of the extracted RNA and not the exact amount. For example, if you have 300ng/ul RNA. You have to pick exactly 3.33ul of your RNA sample which contains 1000ng of RNA. To read about the calculations please read last paragraph of TRIzol RNA Extraction.

Sequence Specific RNA

If you have specific RNA you can pick volume of RNA sample that contains 500ng or 0.5ug of RNA sample.

Choosing Total RNA vs mRNA

mRNA	Total RNA
More sensitive	Less sensitive
More purification step is required	Fewer Purification step
Less amount of RNA is obtained	More quantitative recovery of RNA
Give Absolute quantification	Gives Relative quantification
Less Suitable	More Suitable

From the above table it is clear that although mRNA gives more sensitive results, yet total RNA is recommended as a template for cDNA synthesis because mostly relative quantification is required to study gene expression level.

Primers

There are three primers that can be used in two-step RT-qPCR. Each type of primers has its own advantages and disadvantages. It totally depends upon the choice of experiment that which type of primer should be used. Each primer gives a different starting point to reverse transcriptase for adding dNTPs (Figure 02).

Oligo dT Primer

Oligo dT primers binds to the poly-A tail at 3’ end of mature mRNA. Sometimes, anchored oligo-dT primer is also used in which binds half to the poly-A tail and half to the specific sequence of mRNA which ensures binding of primer to 5’ end of poly-A tail of mRNA.

Random Primer

As the name indicates, random primer can be 6 to 9 bases long but mostly random hexamers are used which bind randomly to the any RNA at many positions thereby providing reverse transcriptase a lot of starting points.

Sequence Specific Primers

When we need to synthesize cDNA for only one gene, we use sequence specific primer which bind only to the specific gene and makes cDNA of only that specific gene.

Sample Type	Advantages	Disadvantages
Oligo dT Primer	Generate full length cDNA only from mature mRNA Used when small amount of RNA sample is available. Anchor ensures that the oligo(dT) primer binds at the 5′ end of the poly(A) tail of mRNA.	Amplify on mRNA containing poly-A tail. Truncated cDNA is obtaind if there is poly A within the gene sequence. Binds only to 3’ end of mRNA.
Random Primer	Anneal to all types of RNA.Used when RNA contains secondary structures.Gives High cDNA yield	Makes cDNA from all RNAs which is sometimes not required. Dilute mRNA signal while qPCR. Truncated cDNA is obtained.
Specific Primer	Only Specific cDNA pool is obtained.Highly sensitive Uses reverse qPCR primer	Synthesis is limited to only one gene.

Nuclease Free Water

Water which is used in this step must be nuclease free otherwise the RNase can degrade RNA template. This can result in low yield of cDNA or it is quite possible that no cDNA is synthesized when using gene specific primers. Nuclease free water comes with commercial kits. You can also treat use the DEPC treated water which is also nuclease free because DEPC degrade all the RNases.

Reaction Buffer

Reaction Buffer of reverse transcription to manufacture cDNA has the following components

Tris-HCL

It maintains the pH upto 8.3 which is necessary for RNA integrity and reverse transcriptase activity.

KCl and MgCl₂

Both provides Mg⁺²and K⁺which act as co-factors of reverse transcriptase and they also help primer binding by reducing negative charge of RNA. In fact, reverse transcriptase use these ions to neutralize the negative charge present on RNA sample and hence it is necessary for RNA dependent DNA polymerase activity of reverse transcriptase.  Lower concentrations of Mg++ lowers Polymerase activity but increases its fidelity and vice versa.

Dithiothreitol

DTT is a reducing agent, so it helps to break bonds (like disulfide bonds) which will loosen the secondard structure of the RNA and facilitate RT enzyme initiation of transcription and processivity.

RNase Inhibitor in cDNA Synthesis

RNase inhibitor which most widely used in cell culture labs is RiboLock RNase Inhibitor which is a 49.6 kDa protein monomer derived from E.coli cells with a cloned gene encoding a mammalian ribonuclease inhibitor. It inhibits the activity of RNases A, B and C by binding them in a noncompetitive mode at a 1:1 ratio. It does not inhibit eukaryotic RNases: T1, T2, U1, U2, CL3 as well as prokaryotic RNases I and H. This inhibitor comes in a solution form and the buffer in which this inhibitor is dissolved is a mixture of 20 mM HEPES-NaOH (pH 7.5), 50 mM NaCl, 8 mM DTT, 0.03% (v/v) ELUGENT Detergent and 50% (v/v) glycerol.

dNTP Mix

dNTP Mix contains premixed aqueous solutions of dATP, dCTP, dGTP. This is better to mix all of dNTPs together to reduce the pipette error and contamination. Reverse transcriptase uses these dNTPs to synthesize cDNA using RNA sample as a template.

Reverse Transcriptase to synthesize cDNA

Reverse Transcriptase is the enzyme that reverse the central dogma of life and can synthesize the DNA from RNA. The RT which is used in labs are commercially available. Mostly, they are mutated structurally to show greater activity. Most widely used RT is moloney Murine Leukemia Virus reverse transcriptase also called M-MuLV RT. It has the following activities:

• RNA-dependent polymerase activity
• DNA-dependent polymerase activity
• RNase H activity specific to RNA in RNA-DNA hybrids

It is obtained by cloning the pol gene from moloney murine leukemia virus into E.coli. Its optimum temperature is 42^OC , can add modified nucleotides and synthesize upto 13kb cDNA strand.
A lot of other RTs are also available commercially whose details can easily be obtained from the official sites or from the manuals of the products.

Procedure

• The protocol for reverse transcription for cDNA synthesis is quite simple. You should just follow the steps.
• Thaw all the components, mix and slightly centrifuge all of them.
• Take 1ug of RNA e.g. 2ul in nuclease free PCR tube (volume can be variable depending upon your RNA concentration).
• Add 1ul of Random Hexamer.
• Add 9ul nuclease free water. The final volume up to this step should be 12ul.
• Keep this mixture into thermal cycler at 65^OC and let the secondary structure of the RNA denature.
• After the incubation, remove the PCR tube and quickly place it on ice to let the primer prime onto the RNA strand.
• Add 4ul 5X Reaction Buffer ,1ul of RiboLock RNase Inhibitor (20 U/µL) , 2ul of 10 mM dNTP Mix and 1ul of RevertAid M-MuLV RT (200 U/µL) RESPECTIVELY.
• Mix and gently centrifuge the PCR tube. The final volume should be 20ul.
• Keep this tube into thermal cycler and set the appropriate protocol.
• Let cDNA be synthesized by reverse transcriptase.
• The final product is single stranded cDNA which is going to be used in the next qPCR step.

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