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Introduction
Polymerase Chain Reaction (PCR) is a versatile and critical reaction in molecular biology. The reaction is a landmark in molecular biology because it has made it possible for scientists to study minute quantities of DNA material. PCR involves the amplification of minute quantities of DNA into large quantities for various molecular studies. PCR operates in a very simple principle that entails a process of DNA polymerization under in vitro conditions. Since PCR is a versatile reaction, advancement in gene technology has led to the improvisation of its operational principles leading to the development of various types of PCR such as real-time PCR, reverse transcriptase PCR, and long accurate PCR among others.
In molecular biology, PCR has many applications and one of the applications is prenatal diagnosis of genetic disorders. PCR has revolutionized the diagnosis of genetic disorders since prenatal diagnosis is now possible given a small sample of fetal tissue. As aforementioned, PCR is a very sensitive method of diagnosis for it entails amplification of minute quantities of DNA into sufficient quantities for diagnosis. Due to its sensitivity and amplification ability, PCR has found a great deal of application in forensic sciences. In molecular biology, the most important function of the PCR is its ability to amplify a specific sequence of DNA material amidst many sequences or fragments that flank it. Although the basic function of PCR is an amplification of DNA material, it is also able to amplify RNA material. For instance, analysis of gene expression levels in embryo development requires the use of mRNA as a product of DNA transcription. To detect gene expression level in terms of mRNA, it requires reverse transcription of mRNA into cDNA for it to undergo a process of polymerization in the PCR. Since the analysis of gene expression level needs reverse transcription of mRNA into cDNA, and subsequent amplification through the PCR process, the whole process is called reverse transcription PCR (RT-PCR).
To analyze gene expression level in plant tissue, this experiment uses RT-PCR. RT-PCR is an appropriate technique of analyzing gene expression as compared to the Northern blot technique because genetic material in plant tissue is very minute and thus requires amplification prior to analysis. Since RT-PCR is a very sensitive method that can detect small quantities of mRNA in plant tissue, this experiment employs reverse transcription and amplification as two critical processes of RT-PCR in determining differential gene expression.
Methods
RNA Isolation
Japonica rice provided the source of plant tissue from which RNA material was isolated. To extract RNA, the root tissue of Japwasa was harvested and homogenized in liquid nitrogen to preserve the integrity of RNA. After obtaining the homogenate, the ethanol extraction method was used to isolate total RNA, and then RB column was used in the purification of total RNA, and finally stored in ice as a purified sample of RNA ready for analysis. Since purified total RNA contained some genomic DNA that could affect RT-PCR analysis, DNase was added to degrade all residual DNA. To ensure that total RNA is pure, a photometer was used to measure absorbance ratio of 260:280nm and absorbance ratio of about 2.0 was considered very pure.
First Strand cDNA Synthesis
Superscript III reverse transcriptase was used in the synthesis of the first cDNA strand providing a volume of 20ul. A master mix that contains RNase, oligo (dT) primers, dNTPs, 5Xfirst strand buffer, and DTT as a reducing agent were prepared. The master mix was added into a purified sample of total RNA and mixed using centrifugation. To carry out the reverse transcription of RNA into cDNA, superscript III reverse transcriptase was added into the overall mix and put into the PX2 thermo cycle for reverse transcription to occur at 500C for a period of 60 minutes.
PCR Amplification
After synthesis of cDNA strand through reverse transcription, cDNA was then amplified through the PCR process. The PCR amplification targeted two specific genes in the cDNA sequence that encodes for actin and separin. Critical for the amplification of the two genes are actin anseparin-specific primers that targeted specific amplification of these genes. PCR master mix contained separin-specific primers, 10 X buffer, DNA polymerase, dNTPs, while sterile water and MgCl2 were prepared in a volume of 25ul. A similar PCR master mix that contained actin-specific primers was also prepared. For control experiments for both genes, an equivalent amount of RNA was used in place of cDNA. PX2 thermo cycle was used to carry out amplification of cDNA sample and RNA sample that acted as a control experiment in the amplification of both actin and separin genes. Thermo cycle was set to the following conditions: denaturation at 940C for 5minutes, annealing at 550C for 30 seconds, extension at 720C for 30 seconds. The PCR underwent 35 repeated cycles of amplification and was finally allowed to undergo an extension step at 720C for 2minutes.
Detection of PCR products
To detect PCR products, electrophoresis was done using 1.0% agarose gel, which was stained with ethidium bromide to enhance the visualization of DNA. Wells were prepared in agarose gel and 10ul of the amplified DNA was loaded into respective wells. Standard marker was also loaded on the adjacent wall as control of estimating various sizes of the amplified DNA fragments. Since the actin and separin sequence of DNA has a molecular weight that ranges from 395bp to 418, agarose gel was expected to show a band that matches with bands of standard marker that fall within the range.
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