Analysis and Amplification of β-actin Gene in Salmon by RNA Extraction and Reverse Transcription Polymerase Chain Reaction (RT-PCR)

INTRODUCTIONβ-actin is a non-muscle cytoskeletal actin ubiquitously expressed in mammalian cells, involving in critical cellular processes such as cell motility, maintaining cellular structure and integrity. [1] It is a common loading control used in many molecular genetics analyses such as Western blotting and the polymerase chain reaction (PCR). Recurrent mutations of the β-actin have been linked to diffuse large B-cell lymphoma, a cancer of the B cell lymphocytes. [2] In this experiment, we aimed to generate multiple copies of β-actin cDNA from extracted salmon RNA, with the eventual purpose of ligating the β-actin gene into a T-vector by TA cloning and transforming the ligated DNA product into bacteria in subsequent practicals. A series of qualitative analysis was performed on to determine the integrity and purity of the extracted RNA from Salmon fish and the DNA from reverse transcription polymerase chain reaction (RT-PCR). Quantitative analysis on the extract RNA was also carried out to detect the degree of β-actin gene expression in Salmon.

Figure 1: Schematic of Experiment

As seen in Figure 1, RNA was first extracted from a Salmon fish sample by the phenol-chloroform extraction. The integrity of the extracted RNA was analyzed by gel electrophoresis, while the Nanodrop spectrophotometer was used to determine the concentration and purity of RNA. The reverse transcription polymerase chain reaction (RT-PCR) was carried out to amplify the β-actin gene and the results were visualized by DNA gel electrophoresis.

Genes are expressed in the cell through mechanisms of DNA transcription and RNA translation. Messenger RNAs (mRNAs) are the intermediate molecules transcribed from protein-coding DNA sequences, and translated into polypeptides by the cellular translation machinery. Since mRNA molecules are more abundant when the complementary DNA sequences are actively transcribed, the level of gene expression can be determined by studying the amount of mRNA transcripts present in the cell at any point of time.[3]

In the first part of the experiment, RNA was extracted from Salmon fish samples by the phenol-chloroform extraction method, commonly used for DNA, RNA and protein extraction. For RNA extraction, a specific reagent, TRIzol, is used to homogenize the sample. TRIzol is a mixture mainly composed of water, phenol, guanidine isothiocyanate and red dye. It breaks down and disrupts cellular components while maintaining the integrity of RNA during homogenization. Guanidinium thiocyanate lyses cells and is used as a denaturing reagent to deactivate RNases and isolate rRNA from ribosomal proteins, because it is a chaotropic agent that disrupts the intramolecular interactions (such as van der Waals forces and hydrogen bonds) in macromolecules.[4] After the liquid homogenate was centrifuged and cell debris removed, the homogenate was incubated at room temperature to allow complete dissociation of nucleoprotein complex and deactivation of RNases.[5][6]

Phenol and water act as organic and aqueous solvents respectively, separating into two phases when chloroform is added. Chloroform further denatures the proteins, such that the proteins are soluble in phenol. The upper aqueous phase will contain nucleic acids (DNA and RNA), and the organic phase will consist of DNA, proteins dissolved in phenol, and lipids dissolved in chloroform. When the pH of the phenol is neutral or slightly basic, the upper aqueous phase will contain nucleic acids (DNA and RNA), which are polar due to their negatively-charged phosphate backbones and hence dissolve in water, which is a more polar solvent than phenol. As the pH of the phenol decreases, the phosphate groups on DNA are neutralized more readily, causing DNA to become less polar and fractionate into the phenol phase, leaving only large DNA fragments and RNA in the aqueous phase. (Acidity also reduces the activity of RNases.) The organic phase will consist of small DNA fragments (<10 kb), proteins dissolved in phenol, and lipids dissolved in chloroform. The interphase between the aqueous and organic phases comprises large DNA fragments and proteins which have large hydrophobic regions that reduce their solubility in both polar phases.[7] The red dye stains the organic layer red and allows the phases to be visually distinct, to facilitate isolation of the aqueous phase by pipetting. Isopropanol was added to the extracted aqueous layer to recover RNA. [8]

Isopropanol disrupts water-RNA interactions and reduces the hydration shell between RNA and DNA, allowing precipitation of RNA which will form a gel-like pellet after centrifugation. DNA, which remains soluble in water, is discarded with the supernatant. The pellet is washed with ethanol to remove any contaminants and dried slightly, to

RNA extracti

on: using TRIzol

reagent

Gel electroph

oresis: Analyze integrity

of extracted

RNA

Nanodrop spectrophotometer: Determine concentrat

ion and purity of

RNA sample

RT-PCR:cDNA

synthesis and

amplification

DNA gel electrophoresis:Visualize RT-PCR results

allow as much leftover ethanol to evaporate from the pellet as possible without the pellet losing solubility. RNA was redissolved in RNase-free water (DEPC-treated water) and incubated at about 60oC for 15 minutes before storage at -80oC.

Subsequently, the concentration (in ng/μl) and purity (OD260nm/280nm and OD260nm/230nm) of RNA samples were determined using a Nanodrop spectrophotometer. The absorption ratios of the RNA sample at different wavelengths are indicative of the purity of the sample due to the different molar absorptivity (also known as molar extinction coefficient, a measurement of how strongly a material absorbs light at a certain wavelength per molar concentration) and optical density (also known as absorbance) of different compounds. Extinction coefficient is an intrinsic property affecting the absorbance of a substance, which refers to the logarithmic ratio of the radiation incident on the material to the radiation transmitted through the material. For instance, the average extinction coefficient of DNA is 0.020 (μg/ml)-1cm-1, while that of RNA is 0.025 (μg/ml)-1cm-1, therefore absorbance changes to a different extent for DNA as compared to RNA, when the wavelength of radiation incident on the sample changes.

The absorption ratio OD260nmm/280nm (also written as A260/280) is the ratio of absorbance of radiation at wavelengths 260nm versus 280nm. OD260nm/280nm is mainly used to check for contamination of RNA sample by genomic DNA, because nucleic acids show a peak in their absorption spectrum at a wavelength of 280nm, thus the spectrophotometer detects RNA and DNA best at that wavelength. RNA shows a higher OD260nm/280nm than DNA due to the higher absorption ratio of uracil compared to that of thymine. Because OD260nm/280nm is approximately 2.0 for pure RNA and approximately 1.8 for a pure DNA sample, an OD260nm/280nm lower than 2.0 indicates contaminating DNA, protein, or phenol in the RNA sample. A higher value is usually due to limitations of the machine or other contaminants.[9, 10] OD260nm/230nm is a secondary measure of purity by detection of organic contamination, such as phenolate ions and thiocyanates. Because of their molecular structure, these contaminants, which include salts and solvents, are detected by the spectrophotometer at a wavelength of 230nm. For instance, phenol has an absorption peak at 270nm and gives a OD260nm/230nm value of 1.2. The OD260nm/230nm for a pure RNA sample is between 2.0–2.2, and a lower value indicates reduced purity due to organic contamination by salts and solvents. A higher value is usually due to errors in the technical operation of the machine. [10] The concentration of RNA samples was computed using the Beer-Lambert law, which relates absorbance (A) to the concentration (c) of the absorbing particle by the formulaA=∈cl, where ∈ and l, the pathlength, are constants.[11]

The integrity and a high level of purity of the RNA sample have to be ensured before RT-PCR can be carried out.

Gel electrophoresis is used to separate different nucleic acid and protein species, identify, purify, quantify their fragments and determine their quality. To perform this technique, an agarose gel with slots is used. As the mechanism is physical rather than chemical, the added sample is easy to recover for storage and reuse. Moreover, agarose gel is relatively safe, recyclable, and gives clear images because agarose is derived from seaweed with agar as the main component. Other types of gel include polyacrylamine, which is more commonly used for analyzing proteins of large sizes (~5 – 2000 kDa), as well as starch, which is a non-toxic but more

opaque medium. The pre-cast gel is formed by adding a hot agarose solution into a shallow container with a “comb” submerged into the gel, removing the “comb” only when the gel has solidified. The DNA or RNA of interest is then placed in the rectangular wells created in the gel by the teeth of the “comb”, and an electric current is run through the gel at neutral pH. RNAs and DNAs are negatively charged due to the phosphates in their structures. During the gel electrophoresis they will migrate towards the anode (the positive end of the gel), with bigger DNA fragments moving more slowly due to greater interaction forces between the fragments and the gel molecules.[12]

In gel electrophoresis, the fluorescent nucleic acid gel dyes essentially determine the quality of images. Ethidium Bromide (EtBr) was commonly used for staining nucleic acids in this technique because of its low cost and relatively sufficient sensitivity, which produced relatively clear images. However, due to its highly mutagenic property and high costs of safe disposal, new dyes with little or no toxicity have been designed such as SYBR Safe, GelRed, GelGreen, etc. For GelRed, it has been proven to be non-mutagenic and non-cytotoxic by various creditable tests [13], with a more environmentally-friendly disposal procedure of direct discard via drainage system or into a trash bin. Images produced are also proven to be of higher clarity than those produced by using EtBr due to better sensitivity: GelRed can detect bands of 0.25 ng while EtBr can only detect bands from 1–5 ng. [14] Also, GelRed is now often used to replace EtBr thanks to its compatibility with the optical setting for EtBr when using ultraviolet trans-illuminators (excitation at approximately 300 nm, emission at approximately 590 nm).[14, 15]

Reverse Transcription Polymerase Chain Reaction (RT-PCR) is a method quite similar to standard PCR, except that it starts with an mRNA instead of a double-stranded DNA. RNA is used as the template for reverse transcription of a strand of complementary DNA (cDNA), using reverse transcriptase and a reverse primer. Next is the similar procedure of amplification of a specific cDNA by the polymerase chain reaction, which includes denaturation, annealing and extension of the product. The whole process is done with a programmed thermal cycler. To analyze the results of RT-PCR, three main methods can be used for quantifying the genes of interest including comparative,

relative and competitive RT-PCR. In this experiment, the qualitative analysis of the results was our main interest, and it was obtained by gel electrophoresis of the RT-PCR product.

Generally, the high concentration of the extracted RNA (784.75ng/μl) has indicated that the results are quite credible. The values of OD260nm/280nm and OD260nm/280nm are slightly above and below the ideal range of 1.8 – 2.0 (2.09 and 1.67 respectively), showing a moderately high purity of the product. However, the RNA and DNA gel electrophoreses show that the RNA is degraded. The DNA electrophoresis further indicates that contaminating nucleic acids are present, and that that primer used to reverse transcribe and amplify the β-actin gene may favour the amplification of another RNA sequence or gene present in Salmon cells. This warns us of experimental errors, which may lead to inaccurate results later on. However, the DNA gel exhibits a rather distinct band at the desired position (~500bp), thus we can still isolate the β-actin gene from the gel, and use it in subsequent experiments.

The results obtained from the gel electrophoreses and RT-PCR will be discussed in detail below.

RESULTSTable 1: Nanodrop spectrophotometer results showing concentration (ng/μl) and purity (OD260nm/280nm and OD260nm/230nm) of extracted salmon RNA samples

Concentration PurityOD260nm/280nm OD260nm/230nm

984.75 ng/μl 2.09 1.67

Figure 3: Photograph of agarose gel after gel electrophoresis of extracted salmon RNA samples (RNA sample 3 was used in subsequent spectrophotometer analysis and RT-PCR)

Figure 4: Photograph of agarose gel after gel electrophoresis of RT-PCR product samples of extracted salmon RNA (DNA sample 3 was the RT-PCR product of RNA sample 3)

DISCUSSIONTable 1 shows that RNA is present at a high concentration of 984.75 ng/μl, validating the reliability of the results. A sufficiently high concentration of RNA is necessary for the spectrophotometer to accurately detect and distinguish absorbance of the sample at different wavelengths. At low concentrations of RNA, the sensitivity of the spectrophotometer is reduced leading to low readings at 230nm, 260nm, and 280nm, such that OD260nm/280nm and OD260nm/230nm no longer show 2:1 ratios (even though the RNA sample may be very pure). Furthermore, a low concentration of RNA may translate to insufficient amounts of RNA to run further tests and double check the results, and increased probability of error.

Table 1 shows an OD260nm/280nm value of 2.09, which is just slightly above the ideal value of 2.0 for optimal RNA purity, and a OD260nm/230nm value of 1.67, which is below the ideal range of 1.8–2.0. This indicates that the RNA sample is pure to a moderately high degree, and the contaminants present are likely of an organic nature, such as proteins, salts or solvents, rather than DNA. Since the ideal value of OD260nm/280nm is approximately 2.0, the measured value of 2.09 falls within the ideal range, indicating negligible levels of contaminating DNA or protein. This implies that the nucleoprotein complex was likely completely dissociated by TRIzol, and the contaminating agents are likely not DNA or RNases. The low OD260nm/230nm value points to organic contamination, likely by residual phenol or guanidinium thiocyanate from the TRIzol used in the nucleic acid extraction step. [9, 10] This may also account for the slightly higher OD260nm/280nm value, as phenol contamination is known to increase the ratio.[10]

DNA ladde

r

DNA sample 3DNA sample 1DNA sample 2

RT(+)

RT(–)

RT(+)

RT(–)

RT(+)

RT(–)

DNA size (bp)

The gel electrophoresis results in Figure 3 show the highest concentration of RNA fragments with sizes around 2.0 kilo base-pairs (kb) – the region of the 18S rRNA band. However, the 28S ribosomal RNA (rRNA) band, located between the 4kb–5kb bands on the RNA ladder, is not visible, and the 18S rRNA band, while brighter and more distinct than the other bands, exhibits a high degree of smear. This implies degradation of the RNA due to presence of contaminating RNases, which break 28S rRNA into smaller pieces of varying lengths that travel further down the agarose gel and are present in insufficient quantity to produce bright, distinct bands. 18S rRNA is also degraded but to a lesser degree, leading to a bright smear that becomes less intense further down the gel. This may not have been reflected in the spectrophotometer results because the RNA sample was diluted prior to spectrophotometer analysis, leading to reduced sensitivity of the instrument. Overall, the spectrophotometer results suggest that the RNA sample is usable for RT-PCR, but from the gel electrophoresis results, we must question the integrity of the extracted RNA, and proceed with caution.

The gel electrophoresis results in Figure 4 show that the most abundant DNA fragment synthesized via the RT-PCR process is 750bp in size. The size of the β-actin gene is known to be 467bp [12], thus the 750bp band does not contain our gene of interest. It is likely that another RNA sequence contains an identical sequence as the one that the primers bind to in the β-actin gene, thus another gene or RNA sequence of 750bp was reverse transcribed and amplified. A smeared band is observed very faintly around 500bp, and this is likely the amplified β-actin DNA, as the electrophoresis results show the DNA fragment size to be slightly smaller than 500bp. The third band is slightly smeared and moderately bright, and consists of nucleic acid fragments smaller than 250bp. This can be attributed to the fact that the RNA is degraded, as shown by the RNA gel electrophoresis results in Figure 3, leading to many shorter RNA fragments of various sizes. The smearing of the two bands closer to the end of the gel can be due to insufficient sensitivity of the gel or the effects of the GelRed UV trans-illuminators. There is also the possibility that cDNA sequences are of different sizes due to random binding between cycles, because the site-specific primer may bind to an identical sequence outside of the β-actin mRNA, if it is present. Nevertheless, the smear is relatively faint, and the ~500bp band, while not bright, is relatively distinct, thus the amplified β-actin DNA can still be isolated and used. The second lane for each DNA sample is RNA sample, prior to reverse transcription, with Taq Polymerase added. It acts as a control to show that any visible band in the first lane (cDNA sample with Taq Polymerase) is due to successful reverse transcription and amplification of that DNA fragment during RT-PCR. The fact the no visible bands are observed in all three second lanes confirms that the RNA sample is not contaminated with β-actin DNA, because the Taq Polymerase can only amplify DNA and not RNA.

Experimental errors likely led to contamination of the extracted RNA. The presence of phenol and guanidinium thiocyanate can be attributed to residual TRIzol in the aqueous phase after phenol-chloroform extraction. The RNA purity could further have been affected by the presence of salts in the Salmon meat, which were drawn together with RNA in the supernatant and thus present in the aqueous phase after phenol-chloroform extraction. Improper micropipetting technique and the difficulty of completely removing the supernatant may lead to TRIzol and salts remaining in the extracted RNA sample. RNases could have been introduced to the RNA sample via several routes. Since TRIzol inhibits RNase activity, the contaminating RNases was likely introduced after the phenol-chloroform extraction. As RNases are present in the air and on virtually all surfaces (including our skin), the RNase could have contaminated the sample during air-drying of the RNA pellet, when the RNA was left exposed to the air. The apparatus used could possibly have been contaminated with RNase, a problem exacerbated by poor lab techniques such as reaching into the laboratory glass bottle with gloved hands to take out the microcentrifuge tubes, or leaving the bottle uncapped or the box of micropipette tips open for too long. Conversations during the experiment likely increased the probability of RNase contamination. Further, both gel electrophoreses displayed the smile effect, which occurs when the electrophoresis is run at too high a voltage, causing the gel to heat up excessively. The gel electrophoresis of the RT-PCR products, in particular, was carried out at 130V to reduce migration time. This leads to irregular migration of the RNA and DNA fragments, resulting in bands that curve upwards at both ends and migrate further in the middle.

To reduce RNase contamination, reduce conversations and wear a mask, and carry out the entire experiment in a more sterile environment, such as in the fume hood. This will reduce the possibility of RNase contamination by RNases in air. Practice proper laboratory techniques such as pouring microcentrifuge tubes onto the cover of the bottle and taking the tubes from there. Contamination by TRIzol and salts in the Salmon meat can be reduced by performing centrifugation for longer durations and at higher speeds, and washing the pellet repeatedly with ethanol after RNA precipitation to remove residual salts.[16] Using 1-butanol for extraction of RNA instead of isopropanol has also been found to reduce the carryover effects of residual TRIzol in the RNA pellet. [17] Further, because the OD260nm/280nm ratio is strongly affected by the pH of the solution, it is recommended to dissolve the RNA pellet in 10 mM Tris·Cl (at pH 7.5) for spectrophotometer analysis, perform necessary dilutions with the same solvent, and caliberate the spectrophotometer with the same solution.[18] To avoid the smile effect, perform gel electrophoresis at a lower voltage closer to 100V, or cool the TAE buffer to prevent the gel overheating.[18]

RT-PCR can be used to assay transcript abundance by relative, competitive and comparative methods, which use the results from the end-point RT-PCR. The detection of gene expression is via the use of fluorescent dyes including EtBr, P32 labelling with the help of phosphorimager or scintillation counting, which quantify a substance via radioactivity or radioactive emission, respectively.[12] In relative RT-PCR, the quantification of product is relative to

the co-amplification of a ‘house-keeping’ gene simultaneously with the gene of interest. This ‘house-keeping’ gene is a gene that is commonly required in normal pathways of the cell, including the one involving the function coded by the gene of interest. Thus, it acts as a control that normalizes the samples, which is used for direct comparison of relative transcript abundances of the samples of RNA synthesized.[19] In competitive RT-PCR, a synthetic “competitor” RNA that can be distinguished from the RNA of interest by a small difference in size or nucleotide sequence is used, but normally a slightly shorter RNA with an almost identical sequence is required for more precise results. A specific amount of competitor RNA will be added to the samples and co-amplified with the RNA of interest using RT-PCR. Finally, a concentration curve of both the competitor RNA and the target RNA is mapped out and compared against one another to find the absolute amount of the RNA of interest in the sample as the concentration of the competitor RNA can be deduced from its known initial amount and number of cycles involved. [20] Lastly, the comparative RT-PCR requires the target RNA to compete for one or a few reagents during the amplification process, with respect to an unaffected standard sequence. By the end of the process, an external standard curve is drawn to determine the target RNA concentration by comparing with the curve or concentrations of the standard sequence.[21] Greater sensitivity is conferred by real-time RT-PCR, which is is a type of RT-PCR that works like real-time PCR, using fluorescent dyes like SYBR Green, or TaqMan probe which emits fluorescence, to track the quantity of amplified cDNA with each cycle – essentially, in real time as the RT-PCR occurs.

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