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Ribonucleic Acid (RNA) is unstable in the nature. Since RNA has many uses, researchers trying to develop method to make it stable and less prone from foreign matter. Researchers have found that RNA can be transfer from RNA to DNA by using an appropriate method. The transfer of the RNA to the DNA is called reverse transcription. Normally, the DNA is transcript from DNA to RNA. But the use of reverse transcriptase, an enzyme found from viral can reverse this transcription process. Reverse transcriptase enzyme can be used in the cloning, gene expression pro-filling and micro array, as well as for mRNA sequencing (RNA-seq).
Nowadays, there are many difference types of commercially available RTase, engineered for specific application. Point-mutant RNAse H minus MMLV RTase (for use in SMART, CapFinder method due to its ability to efficiently add few bases of Cytosine at the end of first-strand of the cDNA in the template independent manner) and also deletion-mutant RNAse H minus MMLV RTase (earlier version of engineered RTase. It is capable of synthesizing long strand first-strand cDNA). The latest breakthrough in RTase technology is their ability to withstand higher incubation temperature (up to 60 C). This is useful to denature mRNA template secondary structure and obtaining full-length first-strand cDNA. These engineered RTase are actually re-engineered MMLV stated before.
Polymerase Chain Reaction (PCR) was developed by Kary Mullis. It is very useful tool to generate abundance of DNA. The application of this tool includes for cloning, sequencing, linkage analysis, mutation analysis, detection of gene expression, mutagenesis of DNA sequences, detection of species-specific sequences from other hosts (such as viral sequences in human heart or in recipients of gene therapy experiments). Besides that, PCR can be used as the initial step in sequence analysis, generating sufficient sample quantities for several subsequent analyses (Towbin., 1995). Besides that, a few researchers believed PCR has been used to characterize, analyze, and synthesize any specific piece of DNA or RNA. The development of PCR has grown exponentially, producing much useful information including diagnosis of genetic variations associated with disease, isolation of pathogens, and genomic structure identification (Rea and O'Sullivan., 2006).
STATEMENT OF PROBLEM
The aim of this research is to identify and find the best method to achieve stable double stranded cDNA (ds cDNA). When mentioned about RNA, the total RNA basically is unstable in nature. In order to generate stable population of expressed mRNA to the ds cDNA, a few methods was developed. The method includes Cap-Finder method, Gubler & Hofman method and also RNA - ligase mediated method. This study will more focus on the Cap-Finder method to archive expressed mRNA transcripts.
SIGNIFICANCE OF STUDY
In this study the total RNA from A549 of human lung adenocarcinoma cell line is used. Later the total RNA will be transcript using SMART scribe RT (Clontech), this process is also known as reverse transcription (RT). As a result, the single stranded cDNA (ss cDNA) is form and subsequent ds cDNA conversion using Advantage 2 Polymerase mix (Clontech) by long range Polymerase Chain Reaction (PCR).
The objectives of this study are:
To generate stable population of expressed mRNAs for archiving and storage purpose.
To gauge for linearity of amplification, the amplified cDNAs will be used as templates in qPCR using 3 genes - VWF, ITPR2 and ACTB, and compared against unamplified samples.
Superscript III should give higher PCR product yield compared to mild-type MMLV RTase
Superscript III should exhibit higher sensitivity over mild-type MMLV RTase
2.1 REVERSE TRANSCRIPTASE
Reverse Transcriptase (RT) was discovered at the middle of twentieth century, by Howard Temin who is interested in understanding on how RNA tumor viruses cause cancer (Varmus., 1987).
Basically, the idea of reverse transcriptase was very unpopular at first as it contraindicated the central dogma of molecular biology which states that DNA is transcribed into RNA which is then translated into proteins. However, in 1970 when the scientists Howard Temin and David Baltimore both independently discovered the enzyme which responsible for reverse transcription, named reverse transcriptase (Encyclopedia., 2010).
According to (MenÂ´endez-Arias., 2008) states that the discovery of RNA-dependent DNA polymerase activity, reverse transcriptase by Howard Temin and David Baltimore leads of modern retro virology.
However, according to (Tarrago-Litvak et al., 2004) states the presence of RT in retroviruses in 1970 by the groups of Howard Temin and David Baltimore should be considered as a marking in the story of biological sciences in the second half of twentieth century.
2.1.2 Natural function in viruses
It is believe that the RT enzyme encoded from the genetic material of retroviruses. After that, the entry of retrovirus into a host cell, reverse transcriptase catalyzes conversion of RNA into DNA. The process enables the virus's genome to be inserted into genome of the host cell, resulting in production of more RNA virus from its DNA. Examples of retrovirus encoding reverse transcriptase include Rous sarcoma virus (RSV) and Human Immunodeficiency virus (HIV), cause of the Acquired Immunodeficiency Syndrome (AIDS) (Biology Encyclopedia., 2010).
A replication enzyme of retroviruses is called reverse transcriptase because it polymerizes DNA precursors. Reverse transcriptase uses the single-stranded RNA in retroviruses as a template for synthesizing viral DNA. This process is unusual on making DNA from RNA is known as reverse transcription. In the reverse transcription, the genetic information was being reverse which from DNA to RNA, rather than from RNA to DNA found in transcription. Because reverse transcriptase is essential for retroviruses such as HIV, it is target of much antiretroviral therapeutics. On the other hand, the RT is also a molecular tool used in cloning of genes and the analysis of gene expression (Varmus., 1987).
2.2 APPLICATION IN MOLECULAR BIOLOGY
Reverse transcriptase, use in the application of Polymerase Chain Reaction (PCR) for the purpose of cloning. In the Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), cloning strategy has been invented for obtaining full-length cDNA clones of low-abundance mRNA (Towbin., 1995).
The widest use of reverse transcriptase in cloning has been use for development of the RNA-dependent DNA polymerase activity to catalyze first-strand cDNA synthesis from RNA templates. Typical protocols for the generation of first- and second-strand cDNA call for the use of reverse transcriptase for first-strand synthesis followed by treatment with RNase H before second-strand synthesis utilizing E. coli DNA polymerase I (Tzanetakis et al., 2005).
2.2.2 Micro array
Other application of RT in molecular biology includes micro array. Micro array is a collection of a large number of short synthetically assembled DNA sequence, called probes, arranged on the substrate. The sequence to be identified, which is typically an RNA is gently washed over the micro array so that it binds (or hybridizes) with the probe having the complementary sequence. The sequence to be tested is usually marked with a fluorescent marker so its location and level of hybridization can be determined. DNA micro arrays are being used to great effect for a number of different applications: they can be used to obtain large scale gene expression data, by making probes corresponding to all the different proteins; micro arrays can also be used to detect point mutations in genes, by making lots of probes each differing by one mutation in one base position and seeing which probes the RNA binds most strongly to (Mahim Mishra).
Besides that, micro array is useful in identifying genetic profiles and has the ability to test and screen of individual genes that may be differentially expressed in different samples. In order to continue to this process, large amount of RNA is needed. Amplification using the PCR helps unlimited degree of amplification, and also faster as well as more cost effective (Nagy et al., 2005).
2.3 DIFFERENT TYPES OF COMMERCIALLY AVAILABLE RTase, ENGINEERED FOR SPECIFIC APPLICATION
2.3.1 Point - Mutant RNAse H minus MMLV RTase
Point-mutant RNAse H minus MMLV RTase has been used in SMART, CapFinder method due to its ability to efficiently add few bases of Cytosine at the end of first-strand of the cDNA in the template independent manner (Bashiardes and Michael Lovett., 2000).
2.3.2 Deletion - Mutant RNAse H minus MMLV RTase
Deletion-mutant RNAse H minus MMLV RTase is an earlier version of engineered RTase. It is capable of synthesizing long strand first-strand cDNA (Bashiardes and Michael Lovett., 2000).
2.3.3 The RTase with an ability to withstand higher incubation temperature
The latest breakthrough in RTase technology is their ability to withstand higher incubation temperature (up to 60 C). This is useful to denature mRNA template secondary structure and obtaining full-length first-strand cDNA. These engineered RTase are actually re-engineered MMLV above (Bashiardes and Michael Lovett., 2000).
2.4 POLYMERASE CHAIN REACTION (PCR)
PCR was believed and developed in the early 1980s by Kary Mullis, who was awarded the Nobel Prizes for Chemistry for the invention in 1933. The PCR is a method for the in vitro amplification of DNA sequences and involves automated cycles of denaturation, annealing and extension (Evans., 2009).
The PCR was developed a few decades ago by Kary Mullis. The PCR has started as a basic science and was used on the study of human disease in clinical research. The advantages of this method derive from its simplicity and automation. PCR amplifies a single copy of DNA sequence into millions of identical copies in a matter of hours, allowing rapid detection of subtle differences in sequence, gene cloning, and mutation analysis, among other use (Towbin., 1995).
PCR is the in vitro enzymatic synthesis and amplification of specific DNA sequences. Two PCR technologies began with the discovery of the first DNA polymerase around 1955. The enzyme was purified end in 1958, but automation and modern PCR technology was not developed until 1983 (Bermingham and Luettich., 2003).
PCR was initially developed by Saiki, Erlich and Mullis at the Cetus Corporation to provide a method for in vitro amplification of specific nucleic acid sequences (Rea and O'Sullivan., 2006).
According to (Dallman., 1998), states that PCR has many application which includes analysis of both DNA and RNA which allowed the dissection of samples either too small or in which the target sequence is present at such low concentration for previous techniques to be of use. In transplantation, PCR has been used as a method for both genotyping and RNA phenotyping.
In addition, PCR can amplifies and clones low abundance DNA sequences from the complex mixtures, even using as little as a single cell as the source of genetic material. Typically, is a highly sensitive method used to amplify deoxyribonucleic acid (DNA) and to identify the presence of specific organisms typically within 5 to 7 days. PCR recently has been applied in the diagnosis of viral uveitis, infectious endophthalmitis, parasitic eye disease, and viral keratitis (Myers., 2009).
Currently, PCR methodology is utilized for cloning, sequencing, linkage analysis, mutation analysis, detection of gene expression, mutagenesis of DNA sequences, detection of species-specific sequences from other hosts (such as viral sequences in human heart or in recipients of gene therapy experiments), questions of paternity, and in identification of transgenic animals carrying a gene of interest. Besides that, PCR can be used as the initial step in sequence analysis, generating sufficient sample quantities for several subsequent analyses (Towbin., 1995).
According to (Bermingham and Luettich., 2003), there are two main types of PCR application, a) Analytical PCR- often from unique samples, e.g. detection of infectious agents in patients samples, genetic analysis, tumor diagnostics, research and forensics; b) preparative PCR, e.g. synthesis of hybridization probes and sequencing templates (Tables 1 and 2).
From the other point of view, PCR is believed to have ability to characterize, analyze, and synthesize any specific piece of DNA or RNA. The development of PCR has grown exponentially, producing much useful information including diagnosis of genetic variations associated with disease, isolation of pathogens, and genomic structure identification (Rea and O'Sullivan., 2006).
2.4.3 Long and Accurate PCR (LA-PCR)
Long PCR (LA-PCR) is the most significant recent advance in PCR technology. It encompasses PCR products in the range 5 kb to a current maximum of around 35 kb. Using phage lambda as a template, (Barnes., 1994) was reported the successful amplification of fragments greater than 20 kb for the first time. He attributed the success of the technique to the inclusion of a small proportion of heat-stable polymerase possessing a proofreading which is 3'-5' exonuclease activity in addition to the heat-stable KlenTaq polymerase (which is an amino terminally truncated Taq polymerase). Similar results have since been obtained using other combinations of proofreading and non-proofreading enzymes, resulting in a 3'-5' exonuclease activity that increased the transcription fidelity at least 10-fold (Taylor and Logan., 1995).
On the other site, researches also believe that LA-PCR allows the amplification of sequences 5 to >20 kb in length. The process where number of nucleotides added before polymerase enzyme dissociation from the template, of the Taq DNA polymerases is limiting. Standard Taq DNA polymerase introduces a mismatched base relative to the sample template on average every âˆ¼104 nucleotides. In a subsequent PCR cycle, the mismatch can lead to Taq DNA polymerase dissociation from secondary template strands, thereby restricting the length of amplicon synthesized. In general, standard PCR is not used to amplify sequences greater than 1-5 kb. In LA PCR, Taq DNA polymerase is combined with a thermostable DNA polymerase that has a proofreading capacity (e.g. Pfu DNA polymerase derived from Pyrococcus furiosus); these combinations can increase processivity 5-10 fold (Evans., 2009).
CapFinder method was described to have possible of addition of any oligonucleotide sequence to the 3' terminus of first-strand cDNA during reverse transcription with the help of the 'template-switching' effect. This effect shows that Moloney Murine leukemia virus reverse transcriptase (MMLV RT) is able to add a few non-template nucleotides (mostly C) to the 3' end of newly synthesized cDNA strand upon reaching the 5' end of the RNA template. At the moment of an oligonucleotide having oligo (rG) sequence on its 3' end (so-called 'template-switch (TS) oligo') is present in the RT reaction, it base-pairs with the attached deoxycytidine stretch. Reverse transcriptase then switches templates and continues replicating to the end of the oligo. As a result of the complementary TS-oligo sequence becomes attached to the 3' terminus of the cDNA (Matz et al., 1999).
The CapFinder method offer a solution on a selective enrichment of full-length mRNA and do not eliminates premature termination products generated in cDNA synthesis and also allows the analysis of limited starting material. This method relies on the terminal transferase and the template-switching activity of the reverse transferase. The addition of cytosine residues to the 3' end of full-length cDNA allows the reverse transcriptase to generate specific anchor sequence complementary to the template-switching oligonucleotide (Schmidt and Mueller., 1999).
CapFinder approach depends on the ability of MMLV Reverse Transcriptase (RT) to add cytosine residues to the 3' end of newly synthesized cDNA upon reaching the 5' end (cap region) of the mRNA. Usually, around 2-4 cytosine residues are added, depending on the reaction conditions. If an oligonucleotide with oligo(G) or oligo (rG) sequences at its 3' most end is included in the incubation medium, its terminal 3-4 G residues could base pair with the 2-4 C residues of the newly synthesized cDNA, thus serving as new template for the RT (template switch). The RT then switches the template and replicates the sequence of the CapFinder oligonucleotide, thus including the complementary CapFinder oligonucleotide sequence at the 3' end of the newly synthesized cDNA (Schramm et al., 2000).
SMART cDNA synthesis kit available from Clontech, utilizes feature of Moloney Murine leukemia virus reverse transcriptase (MMLV RT), its ability to add a few non-template deoxynucleotides (mostly C) to the 3' end of a newly synthesized cDNA strand upon reaching the 5' end of the RNA template. Oligonucleotide containing oligo (rG) sequence on the 3' end, which is called "template-switch oligo" (TS-oligo), will base pair with the deoxycytidine stretch produced by MMLV RT when added to the RT reaction. Reverse transcriptase then switches templates and continues replicating using the TS-oligo as a template. Thus, the sequence complementary to the TS-oligo can be attached to the 3' terminus of the first strand of cDNA synthesized, and may serve as a universal 5' terminal site for primer annealing during total cDNA amplification (14). Recently an improvement to the original procedure was reported (15). Addition of MnCl2 to the reaction mixture after first-strand synthesis, followed by a short incubation, increases the efficiency of non-template C addition to the cDNA and thus results in higher overall yield following cDNA amplification (Mikhail).
In the year of 1996, Clontech was introduced and initially designated 'CapFinder' which later then renamed as the 'SMART' product line. The procedure is based on the ability of the Murine leukemia reverse transcriptase (M-MuLV) to add a few cytosine residues to the 3' end of newly synthesized cDNA on reaching the 5' end of the mRNA. A template-switch oligonucleotide (TSO), containing a terminal 3' poly-G, can then pair with the cDNA 5' poly-C tail, itself becoming a template for reverse transcription (Fernando and Peter., 2010).
2.5.1 Rapid Amplification of cDNA Ends (RACE)
The Rapid Amplification of cDNA Ends (RACE) method is broadly used PCR based method to clone the cDNA ends of mRNA transcripts. RACE incorporate of short piece of DNA as an 'anchor' onto the cDNA end to allow PCR amplification of the cDNA end using a universal primer that anneal to the anchor sequence when combined with a gene specific primer derived from the mRNA fragment. Originally achieved using homopolymer tailing with terminal deoxynucleotidyl tranferase, the DNA anchor was incorporated (Glenn K et al., 2004).
Besides that, the RACE method can be used to isolate cDNA fragment derived from the 5' and 3' ends of genes for subsequent determination of their nucleotide sequence. A wide array of RACE methods was reported previously. Generally, in this method it involves addition of a defined sequence is an 'anchor' to the 5' and 3' ends of first-strand cDNAs which setting and adjusting the stage for subsequent PCR using primer complementary to anchor sequence together with one complementary to a known internal gene specific sequence (Xianzong Shi et al., 2006).
In other site it was believed that RACE was developed to facilitate the cloning of full-length cDNA after a partial cDNA sequence had been obtained by PCR amplification. In the RACE method frequently results in the exclusive amplification of truncates cDNA ends. With the help of incorporated of restriction endonuclease sites into the sequence of the 5' and 3' ends of the PCR primer, the cloning of RACE product can be determined. However, the 5' end of the PCR should be complementary to a known cDNA sequence. In order to achieve the efficiency of the subsequent ligation to the sequencing vector, the choice of restriction site strictly considered (Shigemori., 2005).
Based on the (www.promega.com., reviews 12/09) mentions the RACE is a variation of RT-PCR that amplifies unknown cDNA sequence corresponding to the 3' or 5' end of the RNA. There has two general strategies on the RACE method; a) one amplifies 5â€² cDNA ends (5â€² RACE) and b) captures 3â€² cDNA end sequences (3â€² RACE). In either strategy, the first step requires an enzymes of RT as catalyzes to convert RNA to single-stranded cDNA. For subsequent amplification, two PCR primers are designed to flank the unknown sequence. One PCR primer is complementary to known sequences within the gene, and a second primer is complementary to an "anchor" site (anchor primer). The anchor site may be present naturally, such as the poly(A) tail of most mRNAs, or can be added in vitro after completion of the reverse transcription step. The anchor primer also can carry adaptor sequences, such as restriction enzyme recognition sites, to facilitate cloning of the amplified product. Amplification using these two PCR primers results in a product that spans the unknown 5â€² or 3â€² cDNA sequence, and sequencing this product will reveal the unknown sequence. The information obtained from partial cDNA sequences then can be used to assemble the full-length cDNA sequence.
2.5.2 Virtual Northern Blotting
The northern blotting is part of the gel blotting where the technique is used for visualizing a particular subset of macromolecules such as proteins, or fragments of DNA or RNA. Generally, the process initiate with the electrophoresis method where the molecules are separated. In order to allow the molecules to migrate under influence of electric current, the process is conducted in the gel. Blot them with a nitrocellulose filter which lead to the molecules stick tightly to the filter and will retain their relative positions when flooded with fluid. The filter will be bathed with a solution containing a probe. Probe is a molecule that will combine specifically with the target molecules which carries a mean of visualization, e.g. a radioactive or fluorescent marker (Biology Pages., 2006).
Northern blot is a technique used in molecular biology research to study gene expression. It takes its name from its similarity to the Southern blot technique, named for biologist Edwin Southern. The major difference is that RNA, rather than DNA, is analyzed in the northern blot. Both techniques use electrophoresis and detection with a hybridization probe. The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University. mRNA is extracted from the cells grown in galactose and cells grown in glucose and purified. The mRNA is loaded onto a gel for electrophoresis. Lane 1 has gal mRNA Lane 2 has the glucose mRNA. An electric current is passed through the gel and the RNA moves away from the negative electrode. The distance moved depends on the size of the RNA fragment. Since genes are different sizes the size of the mRNAs varies also. This results in a smear on a gel. Standards are used to quantities the size. The RNA can be visualized by staining first with a fluorescent dye and then lighting with UV. RNA is single-stranded, so it can be transferred out of the gel and onto a membrane without any further treatment. The transfer can be done electrically or by capillary action with a high salt solution. A gal DNA probe is incubated with the blot. The single stranded gal DNA probe binds with immobilized gal mRNA. The blot is washed to remove non-specifically bound probe and then a development step allows visualization of the probe that is bound (Gortner., 1996).
In Northern blot hybridization, DNA is transferred from a gel onto a nylon membrane. This blot is then hybridized with a labeled probe specific to the target of interest. Detection of the probe label affirms amplification of the intended sequence (Evans., 2009).
2.6.1 von Willebrand factor (VWF)
Mutation detection in von Willebrand factor (VWF) gene is quite challenging due to its large size, heterogeneous nature of mutations and the presence of a partial unprocessed pseudogene in chromosome 22 with 97% homology. The VWF gene (178 kb) is located on chromosome 12 and comprises 52 exons and 51 introns, while pseudogene corresponding to exons 23-34 of VWF gene is located on 22q11.22-q11.23, a region relevant for several somatic and constitutional chromosomal alterations (Kasatkar et al,. 2010).
Von Willebrand Factor (VWF) is a glycoprotein circulating in plasma as multimers of increasing size, consisting of disulfidebridge linked dimers of the 225-kDa single-chain molecule. VWF functions also as a transport protein for Factor VIII (FVIII) protecting it from premature activation, degradation. Consequently, the absence or reduction of functional VWF has a dramatic impact on the haemostasis. von Willebrand disease (VWD) is the most common inherited bleeding disorder with a reported prevalence of around 1% in the population, characterised by a reduced level or enhanced functional deficiency of VWF, usually accompanied by decreased FVIII plasma levels and clearance (Stadler et al,. 2006).
The glycoprotein von Willebrand Factor (vWF) stabilizes and transports Factor VIII (FVIII), which is directly involved in the blood coagulation cascade). The purification of vWF is a challenge because it is a multimeric protein with molecular weights ranging from 0.5 to 10 million Daltons (Huang et al,. 1996).
Von Willebrand factor (VWF) is one of largest multimeric glycoprotein in peripheral blood, synthesized and secreted from endothelial cells. It plays a very important role in blood coagulation and primary hemostasis by carrying coagulation factor VIII and initiating platelets adhesion. A mature VWF monomer has 2050 amino acids and contains 5 domains in the order D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK (Wang et al,. 2010).
American Diagnostica states that Von Willebrand Factor (vWF) is a large, multimeric protein (molecular weight of 1,000- 20,000 kD) composed of repeating 270 kD subunits containing 2050 amino acid residues. vWF is synthesized by endothelial cells and megakaryocytes, and is present inmultimeric form in the basement membrane of the subendothelium, in plasma and platelets.
Meanwhile, according to stagos which on Willebrand factor (VWF) is a multimeric plasma glycoprotein of molecular weight as high as 15,000 kDa. It is constituted by "n" identical protomers, each containing two sub-units; each sub-unit has a molecular weight of approximately 270 kDa
MATERIAL AND METHODS
Two types of reverse transcriptase used which are SuperScriptÂ® III RT and Moloney Murine Leukemia Virus (MMLV) RT as the test compounds. Both reverse transcriptases used to synthesize first-strand cDNA from total HeLa RNA. SuperScript III RT is a version of MMLV RT that has been engineered to reduce RNaseH activity and provide increased thermal stability.
SuperScriptÂ® III First-Strand Synthesis System for RT-PCR consisting of Oligo dT, RT buffer (10X), random hexamers, 25 mM MgCl2, 0.1 M DTT, 10 mM dNTP mix, SuperScript III RT, RNaseOUT, E. coli RNase H, DEPC-treated water, total HeLa RNA, Sense Control Primer and Antisense Control Primer.
GenBank Accession Number
3.2 Assay Theory
SuperScriptÂ® III First-Strand Synthesis System for RT-PCR is optimized to synthesize first-strand cDNA from total RNA. The system is stable for 6 months when properly stored at -20 Â°C. RNA targets from 100 bp to >12 kb can be detected with this system. The amount of starting material can vary from 1 pg to 5 Î¼g of total RNA. SuperScript III Reverse Transcriptase is a version of M-MLV RT that has been engineered to reduce RNase H activity and provide increased thermal stability. The enzyme is used to synthesize cDNA at a temperature range of 42.55Â°C, providing increased specificity, higher yields of cDNA, and more full-length product than other reverse transcriptases. Because SuperScript III RT is not significantly inhibited by ribosomal and transfer RNA, it may be used to synthesize first-strand cDNA from a total RNA preparation. cDNA synthesis is performed in the first step using either total RNA with oligo(dT), random primers, or a gene-specific primer. In the second step, PCR is performed in a separate tube using primers specific for the gene of interest. For the PCR reaction, we recommend one of the following DNA polymerases: PlatinumÂ® Taq DNA Polymerase provides automatic hot-start conditions for increased specificity up to 4 kb, PlatinumÂ® Taq DNA Polymerase High Fidelity provides increased yield and high fidelity for targets up to 15 kb, and PlatinumÂ® Pfx DNA Polymerase provides maximum fidelity for targets up to 12 kb.
3.3 Assay Procedure
3.3.1 Primer Design
Gene-specific primer sequence will be obtained from Primer Bank freely available through the internet. The prime will be ordered from specialist Oligo company and once arrived will be suspended in nuclease-free water and kept in -20oC until used.
3.3.2 First-Strand cDNA Synthesis by Reverse Transcription
Total RNA converts into first-strand cDNA, where each component was mix and centrifuge before used. Take about 100 nanograms of total RNA from A549 cell line and mixed with 50 uM one-base anchored oligo-dT, 10 mM of dNTP mix. Then top up with DEPC-treated water to 10 l. Incubate the tube at 65Â°C for 5 min, and then place on ice for at least 1 min.
Next, cDNA Synthesis Mix was prepared by adding- RT buffer (10X), 25 mM MgCl2, 0.1 M DTT, RNaseOUT and SuperScript. III. The tubes of cDNA Synthesis Mix were gently pipetted at 10 Î¼l to mix RNA/primer mixture and were mixed gently. The reactions were centrifuged and incubated for 50 min at 50Â°C. After 50 min, the reactions were terminated at 85Â°C for 5 min and were chilled on ice. The reactions were centrifuged again before adding 1 Î¼l of RNase H to each tube. Then, the cDNA synthesis reaction were incubated for 20 min at 37Â°C and stored at -20Â°C before undergo PCR process. The procedure was repeated 2 more times using reverse transcriptase - Superscript III (Invitrogen) and MMLV.
3.3.3 Polymerase Chain Reaction
Three different transcripts representing low (ACTB), medium (VWF) and high abundance (ITPR2) will be amplified by PCR. In a 0.2ml tube, PCR buffer (1X final concentration), 200ÂµM dNTP mix, 200nM of forward and reverse gene-specific primers and 0.2Âµl (1 unit) of Taq Polymerase will be added, top-up with nuclease-free water and mixed to a volume of 20Âµl. The process will be repeated for each separate reverse transcription reaction representing different amount of total RNA to make a total of 27 PCR tubes. The tubes will then be incubated in thermal cycler with the following protocol.
Program the thermal cycler so that cDNA synthesis is followed immediately by PCR amplification, as follows:
1 cycle: 45-60Â°C for 15-30 min
1 cycle: 95Â°C for 5 min
95Â°C for 15 seconds (denature)
60Â°C for 30 seconds (anneal)
72Â°C for 30 seconds (extend)
1 cycle: 72Â°C for 2 min
3.3.4 Agarose Gel Electrophoresis
To prepare agarose gel, 4 g of agarose gel will be boiled with 200 ml of 1X TAE buffer in a microwave. Once cooled to 55oC, 1Âµl of ethidium bromide will be mix to the molten agarose and poured in the gel caster together with well-forming comb. One the gel harden, it will be transferred into electrophoresis tank filled with 1X TAE buffer. Five Âµl of the PCR reaction will be mixed with 1Âµl of gel-loading buffer and pippetted into the wells. Electrophoresis will be carried out for 45 minutes at 130 volt. After that, the gel will be viewed under UV trans-illuminator to see the signals from the PCR product.
L 1 2 3 4 5 6 7 8 9
SIII VWF 1ng
SIII ITPR2 1ng
MMLV VWF 1ng
MMLV ITPR2 1ng
SIII VWF 10pg
SIII ITPR2 10pg
MMLV VWF 10pg
MMLV ITPR2 10pg
Table 1: The Superscript III and MMLV RTase with difference VWF and ITPR 2 template for 100 ng
The result for RTase 100 ng cannot be obtained because the RTase is broken. In order to make another RTase, it took longer time.
Table 2: The Superscript III and MMLV RTase with difference VWF and ITPR 2 template for 1 ng
As refer to table 2 above, the VWF template with RTase Superscript III and MMLV do not shows any band where no amplifying occur. Meanwhile, for the ITPR 2 template with same Superscript III and MMLV shows there is a band during the gel electrophoresis process. The band indicates that the amplifying of the RTase at the end of the template even it has longer base pair.
Basically, template VWF do not shows any band most probably due to a few factors that need to be considered. One of the factors is extensive secondary structures that occur during the amplifying process. When there is secondary structure in the base-pairing, it may limit its coding potential of the mRNA. Besides that, the gene is not expressed in the sample which in this context is whole blood.
Table 3: The Superscript III and MMLV RTase with difference VWF and ITPR 2 template for 10 pg
Table 3, shows that no band for entire template with the base-pair of RTase Superscript III and MMLV.
The present study is done to evaluate how efficient 2 different RTase (cheap and expensive) to transcribe big targets. The chosen of VWF and ITPR2 transcripts because both have very big and large size of bases. VWF consist about
At 1 nanogram (ng) both RTase manage to successfully transcribed ITPR2 right to the end of the transcript. This is evident in the correctly-sized PCR product amplified. At this point the much cheaper wild-type MMLV is as efficient at transcribing very long transcript as the more expensive SIII.
VWF was not amplifiable in both SIII and wild-type MMLV generated first-strand cDNA could be due to a few factor that needs to be consider which include:
Gene is not expressed in the sample
The transcript is not expressed in the sample used. I used total RNA from human peripheral blood extracted using commercially available kit. To answer this literature search is needed.
5.1.2 Extensive secondary structure
Both reverse-transcriptase failed to transcribe VWF mRNA transcript. The transcript is very long and theoretically could form extensive mRNA secondary structure preventing the enzyme from efficiently transcribing. It is believed that secondary structure in mRNAs can reduce the efficiency of translation and a modest amount of secondary structure can drastically inhibit translation (Kozak,. 1991). One way is to use higher incubation temperature but enzyme stability will be affected.
In the research conducted by (Pelletier and Sonenberg,. 1985) demonstrates that translation efficiency is decreased as the numbers of linkers is increased and support the view that excessive secondary structure at the 5â€² end of eukaryotic mRNA impedes translation. On the other hand, the extensive coding region secondary structures suppress translation to a minimal or to a substantial degree depending on their distance from the initiation codon as in the journal writen by (Liebhaber et al,. 1992).
5.2.3 Degradation of the sample and contamination
The VWF mRNA transcript itself has degraded before transcription begins. Longer transcripts are prone to degradation. This could be due to improper handling of the sample during total RNA isolation, polysaccharide co-precipitation of RNA and RNA contamination. However, this is unlikely as other long transcript - ITPR2 is successfully amplified.
184.108.40.206 Polysaccharide coprecipitation of RNA
Both polysaccharides and polyphenols can interact with nucleic acids by forming insoluble complexes, affecting yield and quality of RNA. This indicated that RNA might be lost due to polysaccharides binding in the homogenate during extraction to reduce yield and quality. A high concentration of LiCl, NaAC and 2.5 volumes pre-cooled ethanol were used to precipitate high levels of polysaccharides. It is effectively removes the polysaccharides without affecting the yield of RNA (Wang et al,. 2005).
In addition, if insoluble starch is converted to soluble polysaccharides and these polysaccharides display physicochemical properties similar to those of RNA. They may co-precipitate and contaminate the RNA during the extraction which may affecting the yield and quality (Asif et al,. 2000).
CTAB (cetyl-trimethyl ammonium bromide)- based extraction protocol for routine isolation of high-quality nucleic acids is used to separate polysaccharides known to interfere with several biological enzymes e.g. polymerases, ligases and restriction endonucleases. They form tight complexes with DNA creating a gelatinous pellet hence the embedded DNA is inaccessible to the enzymes. Sodium chloride facilitated their removal by increasing solubility in ethanol hence preventing co-precipitation with DNA (Muge,. 2009).
Certain polysaccharides are known to inhibit RAPD reactions. They distort the results in many analytical applications and therefore lead to wrong interpretations (Kotchoni et al., 2003). Polysaccharides like contaminants, which are undetectable by most criteria, can cause anomalous reassociation kinetics. Polysaccharide co-precipitation is avoided by adding a selective precipitant of nucleic acids, i.e. CTAB (Padmalatha et al,. 2006).
The addition of high molar concentration of NaCl increases the solubility of polysaccharides in ethanol, effectively decreasing co-precipitation of the polysaccharides and DNA (Fang et al., 1992). Finally, the addition of LiCl selectively precipitates large RNA molecules reducing the amount of RNA present in the final DNA solution (Sambrook et al., 1989). Selective precipitation has an advantage over RNAse treatment in that the RNA is removed and not simply degraded into smaller units (Ribeiro et al,. 2007).
220.127.116.11 RNase contamination
The presence or introduction of RNase during the procedure may result in sample degradation. It is strongly recommended to minimize the potential for RNase contamination by using gloves throughout the procedure, using DEPC-H2O and by treating pipettmen, work area, gel box and gel comb (Qbiogene,. 2002). The most common sources of RNase contamination are hands, dust particles, and contaminated laboratory instruments, solutions and glassware (Agencourt Bioscience,. 2006).
Adapted from "Ten Sources of RNase Contamination" (www.ambion,. 2005) of potential sources of RNase contamination include fingerases, tips and tubes, water and buffers, laboratory surfaces, endogenous RNases, RNA samples, plasmid preps, RNA storage, chemical nucleases and enzymes.
According to the kits provided by Invitrogen, the solution to correct this problem includes additional of control RNA to sample to determine if RNase is present in the first-strand reaction, mixing the recombinant RNase Inhibitor in the first-strand reaction and use of RNaseOUT. Besides that, to prevent RNAse contamination the aseptic conditions of the laboratory should be maintained during experiment.
The result shows the limit of both RTase at very low sample input concentration. At 10 picogram (pg) both enzyme failed to yield any results. However it could be the Taq Polymerase to blame. We used wild-type Taq polymerase during PCR. Substitution with the highly-engineered, more soluble version of Taq such as KlenTaq (Barnes, 1994) could help with the amplification provided the RTase is efficient enough. Sensitive reagents are needed to analyze very low amount of samples such as from clinical biopsies, forensic and paleontological samples.
It is interesting to observe that the cheaper wild-type MMLV could be used to successfully amplify long mRNA targets. This could help researchers with tight budget to run experiments such as full-length cDNA cloning for expression or mutation detections.
Based on the study, the cheaper RTase can be used to amplify very long mRNA transcripts and suitable for downstream applications such as cDNA library construction, full-length cDNA cloning or mutation detections.
Besides that, the first-strand cDNA sample as low as 1ng can be successfully used to amplify very long transcripts.
On the other hand, for very long transcript, it is recommended to run mRNA secondary structure prediction program such as Mfold (Zuker,. 1989) to assess the extent of RNA secondary structure present in the particular transcript.