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The choice of PCR method is related to the design of the study. For example, quantitative PCR or classical PCR can be used to study gene expression at the mRNA and non-coding RNA levels. DNA is used to study genomic changes, gene mutations and polymorphisms, and the choice of PCR can be determined depending on the type of sample, the study method and the sensitivity of the study, the information contained in the gene bank, and so on. The ARMS method is used to detect point mutations in which the primer is designed exclusively based on different alleles. RFLP is used to detect the types of polymorphisms, genotypes, and screening for identified mutations based on differences in fragment size in enzymatic sections of PCR products. The Nested method is used to track the target DNA sequence, which uses two pairs of nested primers. The specificity of this method is very high due to the type of primer. In addition, PCR can be used to identify types of microbial strains, parasites, etc. For example, multiplex PCR has the ability to simultaneously identify several strains of bacteria or parasites in one reaction. In this test, several pairs of specific primers are used simultaneously.

One of the most common mistakes among students is using the term RT-PCR for three completely different tests, cDNA Synthesis, Real Time PCR and RT-PCR. In cDNA synthesis, RNA is used as a template to make the reverse transcriptase enzyme from DNA in a temperature cycle using a primer sequence (oligo dt or random hexamer) from DNA. In classical PCR and qPCR, cDNA is used as a template and will be amplified from a gene with a specific primer in several temperature cycles. The difference between the two PCRs is that qPCR uses a special thermocycler that can detect the reflected fluorescent signal and ultimately quantify the results. But in classical PCR, the results are reported semi-quantitatively, in which PCR products can be reported quantitatively after separation in gel electrophoresis with special software.

Freezers with a temperature of -80 °C are a good option for long-term storage of biological materials. This temperature prevents the breakdown of nucleic acids, proteins and many biological molecules. In cases where a temperature of -80 °C is not available or the conditions for sending the sample with dry ice are not possible, the samples can be immersed in aqueous sulfate salt solutions. A commercial, ready-to-use compound called RNALater is available that can be added to tissues isolated from an organism or cell. Solid tissues can be stored at RNALater at room temperature for one week. For long-term storage, samples containing this solution should be stored at -20 °C. To move and transfer frozen samples, it is necessary to maintain a negative temperature of -80 °C using industrial dry ice and transfer the samples to the desired laboratory immediately.

In the study of methylation, the basis of the performance of techniques based on biochemical changes by bisulfite is that non-methylated cytosine are converted to uracil with no modification on methylated cytosine. Therefore, depending on the presence or absence of methyl on cytosine, bisulfite causes special changes in DNA that can be evaluated by PCR, sequencing and microRNA methods. There are several methods available today to study DNA methylation analysis. However, no specific method is known as the “original” technique. Each method has advantages and disadvantages. Methods in which commercial kits can be used to detect methylation are known as the best methods for bisulfite treatment.

Depending on the technique, the quality required for the extracted RNA and DNA varies. High quality RNA is required in a variety of sequencing methods such as NGS, while a medium quality sample is sufficient for the PCTR real-time process. For this purpose, it is necessary to be informed about the quality of the extracted sample before doing the work. In the initial examination, the extracted sample is separated using 1.5% agarose gel. In a quality sample of DNA the corresponding band is seen on top of the gel. In RNA samples, 3 fragments of RNA, 18 S, 28 S, 5.8 S are separated into 3 separate bands. The presence of a smear in the gel indicates RNA degradation. In another method, a Bioanalyzer is used to accurately determine the quality of the extracted RNA, which is displayed as the RNA Value. In degraded RNA samples, the peak height for 28S and 18S rRNA peaks decreases, and in quality samples, peaks will be observed separately. The closer the RNA number is to 10, the higher the quality of the sample.

There are several software such as UCSC, Primer3, MEDUSA, Oligo7 for primer design, which has facilitated the primer design process for the user. After designing in Primer3 online software, primers should be checked on the Primer BLAST page on the NCBI website (http://www.ncbi.nlm.nih.gov/tools/primer-blast) for checking specificity. In general, the following points should be considered in the design of primers: – Its length should be between 18 and 30 nucleotides, – It is better that the GC content is between 50% and 60% (increasing the amount of GC content leads to an increase The connection temperature will be followed by an increase in the strength of the hybrid and the primer dimmer) – There is no significant difference between the connection temperatures of the two leading and back primers (in optimal conditions this difference should be between negative 5 and 5) -Absence of complementary regions in a primer or different primers, especially at the end of 3 primers (to create a strong bond, the 3 ends must be one of the G or C nucleotides) – Tm primers should preferably be designed between 58-60 ° C and the maximum difference between Tm and two primers 3 degrees – Primer should be designed from stable regions (less likely to mutate) of target DNA Replication is done from cDNA that lacks introns, genomic DNA will not replicate.