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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 12  |  Issue : 4  |  Page : 170-181

Transcriptional regulation of protein S gene


1 Faculty of Health and Wellbeing, Sheffield Hallam University, Sheffield, UK
2 Department of Biosciences and Chemistry, Sheffield Hallam University, Sheffield, UK

Date of Submission11-Jun-2018
Date of Acceptance11-Jun-2018
Date of Web Publication11-Nov-2019

Correspondence Address:
Maha Dawood Jaffarali
Faculty of Health and Wellbeing, Sheffield Hallam University, Sheffield
UK
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/HMJ.HMJ_49_18

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  Abstract 


Background: Protein S (PS) is a known Vitamin K dependent plasma glycoprotein that is produced in many human tissues including the liver. Furthermore, it has an important function in the coagulation cascade as an anticoagulant cofactor when it binds active protein C. The deficiency of PS may lead to the development of deep vein thrombosis. There are many transcription factors which play an important role in the regulation of the transcription of PS. One of these regulatory factors is the liver specific transcription factor hepatocyte nuclear factor 1 (HNF1), a binding site which is located in the 638 base pair promoter area at the 5' end of the PS gene. HNF1 and PS is not well understood and to confirm the importance of HNF1 in regulating expression of PS small interfering ribonucleic acid (siRNA) which will knockout the HNF1 gene expression can be used, and the effect on PS expression monitored. The levels of expression of HNF1 and PS can be determined by real time polymerase chain reaction. Materials and Methods: Preparation of total ribonucleic acid for complementary DNA synthesis, Evaluation the quality of HepG2 ribonucleic acid by agarose gel electrophores is, Real time polymerase chain reaction, Acrylamide gel electrophoresis. Results: HepG2 rRNA bands shows a good quality of RNA expression, Analysis of the total RNA quantity can be determined by spectrophotometric analysis, The best housekeeping genes were UBC and YWHAZ therefore, there has been used as a reference for the primers samples in efficiency test. HNF1 and PROS1 shows a good expression but with low efficiency which is not required to carry further gene knockdown experiment, SYBR green dye is less sensitive than TaqMan because it presents a non specific detection (primer dimers) were all dsDNA products are detected including primer dimers, contaminating DNA, and PCR product from mis annealed primer. Conclusion: The primer pairs are not efficient to carry further gene knockdown experiment and required to design another HNF1 and PROS1 primers. The efficiency of the PCR should be 90%-100% meaning doubling of the amplicon at each cycle. This corresponds to a slope of -3.1 to -3.6 in the Ct vs log template amount standard curve. To obtain accurate and reproducible results, reactions should have efficiency as close to 100% as possible.

Keywords: Protein S, HNF1, housekeeping genes


How to cite this article:
Jaffarali MD, Hall A. Transcriptional regulation of protein S gene. Hamdan Med J 2019;12:170-81

How to cite this URL:
Jaffarali MD, Hall A. Transcriptional regulation of protein S gene. Hamdan Med J [serial online] 2019 [cited 2020 Feb 24];12:170-81. Available from: http://www.hamdanjournal.org/text.asp?2019/12/4/170/270678




  Introduction Top


Protein S (PS) is an anticoagulant Vitamin K-dependent plasma gamma-carboxylated glycoprotein of 75 kD molecular weight consisting of 635 amino acids residues. PS is mainly synthesised in the liver but is also made by endothelial cells, testicular Leydig cells [1] and megakaryocytes in culture.[2] Total PS concentration in the human plasma is approximately 0.35 μM: 38% as free PS form and 62% as PS–C4 binding protein (C4BP) complex form.[3],[4] Moreover, the PS genetic locus, PROS, consists of two genes which are located on chromosome 3 but on different sides of the centromere: an active alpha PS gene (PROS1) at q11.2 and an inactive beta pseudogene (PROS2) at p21-cen.[5] The length of the PROS1 gene is more than 80 kilobases and contains 14 introns and 15 exons. Exons I–VIII encode protein segments that are homologous to Vitamin K-dependent clotting proteins and are bounded by introns whose position and type are identical with other members of this protein family.[6] PS inhibits blood coagulation by serving as a non-enzymatic cofactor for activated protein C in the protein C anticoagulant pathway in inactivation of the pro-coagulant factor Va (through proteolytic cleavage at three sites in the heavy chain, Arg-306, Arg-506 and Arg-679)[7] and factor VIIIa in the coagulation cascade.[8] [Figure 1] illustrates the role of PS in the coagulation cascade.
Figure 1: (a-c) Coagulation is initiated through the interaction of tissue factor, upregulated by inflammatory stimuli, with Factor VII, which generates activated Factor VIIa. The tissue factor–Factor VIIa complex then catalyses the conversion of Factor X to Factor Xa, leading consecutively to the activation of Factor V and the conversion of prothrombin to thrombin (FIIa). Thrombin then directly causes clot formation by cleaving fibrinogen to fibrin and also activates FXIII. (d) Inhibition of coagulation cascades occur through inactivation of FVa by protein S and activated protein C generated by the thrombomodulin–thrombin complex. Excess thrombin is compensated by direct inhibition by anti-thrombin III via formation of the thrombin–anti-thrombin III complex[9],[10]

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This anticoagulant protein is essential to maintain blood haemostasis for the supply of oxygen and nutrients to tissue cells and for the removal of toxic by-product from metabolism. Hereditary (major causes and it is an autosomal dominant trait) or acquired deficiencies of PS can lead to many disease states such as arterial thromboembolism and deep vein thrombosis with the possibility of producing lung emboli. Furthermore, PS acts as a cell adhesion protein through interactions with the extracellular matrix and cell plasma membranes.[11] Moreover, this protein has an apoptotic function when it binds to phospholipids via the carboxylated GLA domain.[12],[13] There are many transcription factors involved in the expression of the PROS1 gene and one of these factors is hepatocyte nuclear factor 1 (HNF1) which exists in liver β-cells.[14] HNF1 is a member of the nuclear receptor super family. It not only regulates the expression of PROS1 but also many liver genes by binding to particular sites in the promoter region of those genes.[15] Based on these sites, HNF1 binds DNA sequences similar to GGTTAATAATTACCA.[16] However, the nature of HNF1 in regulating PROS1 transcription is not deeply understood at the molecular level. The relationship between PROS1 gene expression and the transcription factor HNF1 can be studied by the use of small-interfering ribonucleic acids (siRNAs), which are made of short (21–23 nucleotides) double-stranded mRNA (interference RNA) molecules, for knockdown of target gene expression. The siRNAs can be sliced from longer double-stranded RNA (dsRNA) by an ATP-dependent ribonuclease called DICER. DICER is a member of the RNase III family of dsRNA-specific endonucleases. The siRNAs assemble with yet-to-be identified proteins of an endonuclease complex. Studies in eukaryotic cells suggest that the siRNA/protein complexes are then transferred to a second enzyme complex, the RNA-induced silencing complex (RISC), which contains an endoribonuclease that is distinct from DICER, termed slicer. RISC uses the sequence encoded by the antisense siRNA strand to find and degrade mRNAs of complementary sequence.[17] [Figure 2] demonstrates the general siRNA mechanism.
Figure 2: The mechanism of small-interfering ribonucleic acid action. Long double-stranded ribonucleic acid is introduced to a cell. Once it is in the cytoplasm, the dicer enzyme cleaves the double-stranded ribonucleic acid into small-interfering ribonucleic acid with 2 nucleotide 3' overhangs. Following cleavage, ribonucleic acid-induced silencing complex loads and unwinds the small-interfering ribonucleic acid, and binds to the complementary target mRNA which is subsequently cleaved by the ribonucleic acid-induced silencing complex. Following cleavage, the ribonucleic acid-induced silencing complex disassembles and is ready to generate another small-interfering ribonucleic acid for cleavage of additional mRNA[18]

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Using siRNA molecules will inhibit the formation of HNF1 protein by cleaving the target mRNA, and if the expression level of PROS1 is reduced, this means that HNF1 is a regulatory factor for the transcription of PROS1.[19],[20],[21] The expression of these genes (HNF1 and PROS1) may be detected by real-time polymerase chain reaction (RT-PCR) using the SYBR green dye chemistry which binds to double-stranded DNA (dsDNA) and emits light when excited. However, it is less stable and less instrument-compatible than EvaGreen dye.[22] Usually, RT-PCR is a sensitive, efficient and reproducible method to detect and amplify specific gene expression in an organism to be studied. This experiment studies the expression efficiency of PROS1 and HNF1 in relation to housekeeping genes by RT-PCR.[23]


  Materials and Methods Top


Preparation of total ribonucleic acid for complementary DNA synthesis

Growing of HepG2 cell culture

Before starting splitting the cell culture (to have sufficient mRNA for the experiment), the working area in laminar-flow hood and all needed materials were sterilised with barrycidal 36 disinfectant (to kill any present microbes and to avoid contamination of the cells). A flask containing a layer of HepG2 (human hepatocellular liver carcinoma cell line) cells grown in culture for 7 days was collected by rinsing the cells gently for 2 min with 10 ml Dulbecco's phosphate buffered saline (D-PBS) solution (1X D-PBS liquid with 0.1 g/L CaCl2 and 0.1 g/L MgCl2) and the D-PBS was discarded to remove any dead cells. Then, the adherent monolayer of cells was removed from the culture surface by incubating the culture with 5 ml 1X trypsin-EDTA solution at 37° C, 5% CO2 for 10 min. After 10 min, the flask was removed from the incubator and tapped hard against the palm of the hand to detach the remaining adherent cells. The separated cell was mixed with 5 ml Dulbecco's modified Eagle medium (DMEM: 4500 mg/L glucose GlutaMAX™II Pyruvate with addition of 10% GIBCO foetal bovine serum, 1% penicillin-streptomycin and 1% amino acid and 2 mM L-glutamine) to inactivate trypsin-EDTA by balancing the pH and to avoid any damage to cell membranes that may be caused from long exposure to trypsin-EDTA. The homogenised mixture (5 ml DMEM and 5 ml trypsin EDTA) was centrifuged in 25 ml tubes at 20° C (speed: 1000 rpm; REV/MIXX1000) for 5 min. Then discarded and the cells mixed well with 15 ml DMEM by pipetting (10 ml sterile plastic pipette) up and down to get rid of any cell clumps. Finally, 5 ml of the mixture was added to each tissue culture flask (IWAK 3 × 3110-075) with double seal cap containing 25 ml of DMEM.[24] The ratio of splitting the cells was 1:6 (to accelerate the growth of the cells in 7 days) and cell growth (for one of the cell line) was monitored with light microscopy at day 4 and day 7 as shown in [Figure 3]a and [Figure 3]b, which indicates that the number of normal and healthy cells increased after 1 week and were ready to extract total RNA from different cell lines (three different pellets of HepG2 each time from different cell lines).
Figure 3: Healthy and normal growth of HepG2 cells in day 4 as well as increasing the number of cell in day 7 under the light microscope resolution at 10 × 0.22 (XLICAP Still Image Capture). (a) Cell growth after 3 days, (b) cell growth after 6 days

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Extraction ribosomal ribonucleic acid using GenElute™ Mammalian Total Ribonucleic Acid Miniprep Kit (Sigma-Aldrich)

Seven different samples of HepG2 were collected after 7 days of growth (different days and time). HepG2 cells were collected by following the mentioned above splitting cell procedure; however, after the centrifugation step, the pellet was centrifuged again with 20 ml D-PBS at same temperature and speed as mentioned above (1.1) for 5 min. After 5 min, the cells (in 40 ml tube) were kept on ice (to stop any enzymatic activity) or stored at −70° C (for later use). To extract RNA, the D-PBS was discarded and the pellets were mechanically disrupted by vortexing them for 1–2 min. The next steps are based on Chomczynski and Sacchi,[25] single-step method of RNA isolation by acid. The tube (containing cells) vortexed again for 2 min with 250 mL of lysis solution/2-mercaptoethanol (2-ME) mixture (10 mL 2-ME and 1 ml lysis solution) to break protein disulphide bonds (to ensure complete cellular disruption) and inactivate RNase enzyme. The lysed cells were filtered in a GenElute filtration column (2 ml tube with blue insert) and centrifuged for 2 min at speed of 16,000 ×g (maximum speed) to get rid of the debris and shear the genomic DNA. 250 mL of 70% ethanol solution was mixed with the lysate, 700 mL of the lysate/ethanol mixture was transferred to a 2 ml tube (GenElute binding column) and the tube was centrifuged at 16,000 ×g for 1–2 min. The column was washed once with 500 mL of wash solution 1 (centrifuged at maximum speed for 15 s) and twice with 500 mL of wash solution 2 (containing ethanol) and centrifuged at maximum speed for 15 s and then for 2 min, respectively. The column was dried from ethanol by centrifugation at maximum speed for 1 min. To purify 45–90 mL of total RNA, binding column transferred to 2 ml collection tube and centrifuged with 50 mL of the elution solution at maximum speed for 1 min. The total RNA was stored at −70° C until use.

Evaluation the quality of HepG2 ribonucleic acid by agarose gel electrophoresis

Preparation of 1 L of 1X Tris-Borate–EDTA Buffer

In the fume hood, 107.80 g Tris base, 55 g colourless crystal boric acid (toxic if inhaled) and 7.44 g disodium EDTA dehydrate were dissolved in 800 ml of distilled water. After that, the pH was checked and adjusted to 8.3 (base) with boric acid solution and the final volume was brought to 1 L with distilled water (10X TBE buffer). Then, 100 ml of 10X TBE was diluted with 400 ml deionised water (dH2O) to have total volume of 1000 ml (1X TBE).

Preparation of agarose gel

In 250 ml Erlenmeyer flask, 0.3 g of agarose (Bioline) was suspended in 30 ml 1X TBE buffer. The flask was placed into the microwave oven and the solution was boiled for 30–35 min and until all of the small solid agarose particles are dissolved. In addition, the microwave oven was stopped and the flask was swirled gently every 15 s to help dissolve the agarose faster. The molten 30 ml gel solution was cooled to 60°C before pouring the gel in the gel electrophoresis tray (≈7 × 10 cm). After pouring the gel, 7-well comb (≈3 mm) was placed in the gel with addition of 3 mL of ethidium bromide (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide) to stain nucleic acid which will fluoresces bright reddish-brown when exposed to ultraviolet (UV) light. The gel was left at room temperature for 15–20 min to cool and solidify.

Running ribonucleic acid molecules in the horizontal agarose gel electrophoresis

Seven tubes were labelled from 1 to 7 and each tube contained a mixture of 7 mL HepG2 RNA (different samples) – 3.5 mL loading buffer solution (10 mM Tris-HCL, pH 7.5; 50 mM EDTA; 10% Ficoll ® 400; 0.25% bromophenol blue; 0.25 xylene cyanol FF; 0.4% orange G) and 3.5 mL total RNA. After centrifugation for 30 s, the comb was removed from the gel and 7 mL of the RNA-loading buffer (to stain the RNA) was running in seven gel electrophoresis wells at 115 V and 400 mA for 15 min. The gel was denatured and the RNA molecules moved in the gel according to their size (smaller migrate faster than the bigger one). After that, the gel was exposed to UV light (Bioimaging System UV Light) in Epi Chemi II Darkroom to visualise the RNAs and to check their quality. [Figure 4] demonstrates UV images for ribosomal RNA (rRNA) bands (18–25 s in size) in agarose gel for different samples of HepG2: 1–7.
Figure 4: Representative digital image of an ethidium bromide stained agarose gel. Seven different samples of HepG2 total ribonucleic acid which was isolated using the GenElute™ Mammalian Total Ribonucleic Acid Miniprep Kit. Lane 1, 5 and 7: ribosomal ribonucleic acid bands between 18s and 28s in size (red arrows show a clear 2 ribosomal ribonucleic acid bands). Lane 2-4 and 6: smear of ribosomal ribonucleic acid (which indicates degraded RNA). Approximately 3.5 mL of each ribonucleic acid stained with 3.5 loading buffer was electrophoresed on a 1% denaturing agarose gel and stained with ethidium bromide

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Complementary DNA synthesis by ImProm-II™ reverse transcription system kit (Promega) for polymerase chain reaction amplification

In a 1.5 ml Eppendorf tube, to 10 mL of HepG2 RNA (one of the quality HepG2 samples from different HepG2 cell lines 1, 5 and 7 as shown in [Figure 3]) was added 6 mL nuclease-free water, 16 mL ImProm-II™ 5X reaction buffer, 8 mL MgCl2 (final concentration 1.5–8.0 mM), 4 mL dNTP mix (final concentration 0.5 mM each dNTP), 2 mL recombinant RNasin ® ribonuclease inhibitor and 4 mL ImProm-II reverse transcriptase. This mixture synthesised a total volume of 50 mL complementary DNA (cDNA) (transcription of single-stranded RNA to dsDNA in 40 min for combined RNA and random primers) which provided sufficient to template for 24 RT-PCR wells (each well contains 2 mL cDNA excluding negative control wells). The tube was placed in controlled-temperature heat block equilibrated at 25° C for 5 min to let the primers to anneal. For extending phase, the tube was incubated at 45° C for 30 min. Finally, the reaction of reverse transcriptase enzyme was inactivated by heating the tube at 70° C for 5 min and quickly chilled on ice or stored in −70° C.[26]

Real-time polymerase chain reaction

Designing of hepatocyte nuclear factor 1 and PROS1 primers

As the first step, DNA primers for HNF1 (locus NM_000545; 3249 base pair [bp]) and PROS1 (locus NM_00313; 3309 bp) were chosen from basic local alignment search tool with a product size of 60–120 bp.[27] Both primer sets were designed by online ready software programme SGD™ Pages Database (seq.yeastgenome.org/cgi-bin/web-primer) based on the melting temperature (Tm: 57° C–60° C) for previously designed primer sets for the housekeeping genes [Table 1] which based on the G:C bp content (higher GC content will increase the melting temperature due to three hydrogen bonds between G and C which will make them more stable and need more energy or heat to be broken).[28],[29],[30] Then, the designed primers have been ordered from MWG Company. [Table 2] illustrates the primers (HNF1 and PROS1) data that used for the analysis of mRNA expressions by RT-PCR.
Table 1: Two designed DNA primers: Hepatocyte nuclear factor 1 and PROS1 that used in this study

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Table 2: Designed primers that used to analyse mRNA expressions by real-time polymerase chain reaction

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Analysing the expression (checking melt curves) of housekeeping genes by real-time polymerase chain reaction

Housekeeping genes samples preparation

All non-biomaterials (tips, distilled water, micro-pipettes) were placed in the pre-PCR hood and exposed to UV light for 5 min to kill any living molecules. Eight Eppendorf tubes were labelled from 1 to 8 and each of them had a mixture of 59.5 mL double distilled water (ddH2O), 87.5 mL SYBER Green-490 (container covered with aluminium foil due to its sensitivity to direct light) and 7 mL of each housekeeping primer (total of 14 primers up-steam and down-stream): GADPH 1, BACTIN1, B2M1, HPRT1, RPL13A, SDHA, UBC and YWHAZ, respectively. In a plastic PCR plate, 45 PCR wells were divided into eight rows (A–H) and five columns (2–6). The first column was ignored to avoid evaporation of the liquid contents. In addition, the last two columns (5 and 6) were set as negative controls and each of them (total volume 25 mL) will contain 10.5 mL ddH2O, 12.5 mL SYBR-490 and 1 mL of forward housekeeping gene primer and 1 mL reverse one. All columns had contained 8.5 mL H2O, 12.5 mL SYBR-490 and 2 mL cDNA, but first three rows have their specific 2 mL primer (forward and reverse), i.e. row A: GADPH 1, row B: BACTIN1, row C: B2M1, row D: HPRT1, row E: RPL13A, row F: SDHA, row G: UBC and row H: YWHAZ. [Figure 5] demonstrates the location of each housekeeping gene primer in PCR wells as well as the location of the negative controls.
Figure 5: The order of each housekeeping gene primer in real-time polymerase chain reaction wells. Columns 5 and 6 present wells containing negative controls (shaded in green), which consist of 10.5 double distilled water, 1 mL of each primer (forward and reverse) and 12.5 mL SYBR-490. Each row contains 1 mL of each primer (forward and reverse) columns 2–4 (and 2 mL complementary DNA, 8.5 mL double distilled water and 12.5 mL SYBR green); A: GADPH 1; B: BACTIN1; C: B2M1; D: HPRT1; E: RPL13A; F: SDHA; G: UBC; H: YWHAZ

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Before running the micro-wells in the iCycler (BioRad program; Lindsey Bunn protocol – SYBR-490), the wells were sealed with a plastic wrapper. The total time for PCR was 2½ h and the PCR programme consisted of four cycles: Cycle 1 was 95°C for 15 min, followed by Cycle 2 which has three steps. First step was 95°C for 15 s to ensure that all dsDNA and primers are denatured (disruption of hydrogen bounds between complementary bases of the DNA strands). In the second step, the reaction temperature was decreased to 61°C for 15 s to anneal the primers with the single-stranded DNA template (The temperature at this step depends on the melting temperature of the primers), and in the third step, temperature increased up to 72°C for 30 s to expand the primers; steps 1–3 were repeated 45 times. Cycle 3 took 30 s at 95°C for denaturing phase as described in the first step (Cycle 1 and 2) followed by the final cycle step of 50°C for 30 s.[31]

Detecting the expression for hepatocyte nuclear factor 1 and PROS1 oligonucleotides by real-time polymerase chain reaction

The same above PCR procedures were performed for HNF1 and PROS1 primers including six housekeeping gene primer sets: B2M, HPRT1, RPL13A, SDHA, UBC and YWHAZ (well expressed), which were selected to be run again with the designed oligonucleotides. The RT-PCR repeated again for the same primers as shown in [Figure 4]. [Figure 6] demonstrates the location of the primers in the PCR wells.
Figure 6: The order of each housekeeping gene primer in real-time polymerase chain reaction wells. Columns 5 and 6 present wells contain negative controls (shaded in green), which consist of 10.5 double distilled water, 1 mL of each primer (forward and reverse) and 12.5 mL SYBR-490. Each row contains 1 mL of each primer (forward and reverse) columns 2–4 (and 2 mL cDNA, 8.5 mL double distilled water and 12.5 mL SYBR green): A: Hepatocyte nuclear factor 1; B: PROS1; C: B2M1; D: HPRT1; E: RPL13A; F: SDHA; G: UBC; H: YWHAZ

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Testing the 100% efficiency (quality control) of hepatocyte nuclear factor 1, PROS1, YWHAZ and UBC by real-time polymerase chain reaction

Six samples were prepared from each cDNA (HNF1, PROS1, YWHAZ and UBC) in 24 PCR wells. The second column (row B2–E2) has 2 mL (neat volume) from each primer B2: HNF1, C2: PROS1, D2: YWHAZ, E2: UBC, respectively, and columns 3–6 (B3–B6, C3–C6, D2–D6 and E2–E6) have same samples (2 mL each) in the same order but with different concentrations: i.e. 1:10, 1:100, 1:1000 and 1:10000. Each well in the last column (B7, C7, D7 and E7) set as negative control which has only 2 mL of ddH2O in place of the cDNA.[32] [Figure 7] presents the order for each primer in RT-PCR wells.
Figure 7: Testing the %efficiency for the primers and two housekeeping genes. The order of each primers for real-time polymerase chain reaction as follows: Row1 (B2–B7): hepatocyte nuclear factor 1, Row 2 (C2–C7): PROS1, Row 3 (E1–E7): YWHAZ and Row 4 (F1–F7): UBC, The last column (7) contains only double distilled water as negative control for all the primers and the first column (2) has a neat volume for each gene

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PCR efficiency (E) for all the samples were calculated by follow equation: E% =10(−1/slope) − 1.[33],[34],[35],[36] The slope can was determined from plotting the number of threshold (Ct) value via concentration [Table 3] and [Figure 8].
Table 3: Threshold cycle value for hepatocyte nuclear factor 1 (B2-B6, pseudogene 1 (C2-C6) and YWHAZ (D2-D6)

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Figure 8: Calculations of polymerase chain reaction efficiency by determine the value of the Slope (Y axis) and Ct (X axis) from the concentration for each sample

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Acrylamide gel electrophoresis

Two acrylamide gels were prepared and the first wells in each of vertical acrylamide gel (2 ml of 40% acrylamide/bis-acrylamide solution mixture [19:1], 8 ml 1X TBE buffer, 150 mL 10% APS, 15 mL 99% 3N–TD N, N, N′, N′-tetramethylene diamine) was loaded with 6 mL (1st gel) and 8 mL (2nd gel) of 25 bp DNA ladder (is suitable for sizing dsDNA from 25 to 500 bp) stained with 1 mL blue orange loading dye (Promega). Sixteen amplified samples (5 mL from each samples stained with 1 mL blue orange loading dye [Promega]) by RT-PCR and their negative controls loaded in two gels (8 wells each). The first gel was divided from 1 to 8 wells (excluding the ladder) which contained eight samples: 1–3: UBC, 4–5: negative controls for UBC, 6–7: YWHAZ and 8: negative control for YWHAZ. The other eight samples were loaded in the second gel and the wells were divided from 9 to 16 as follows: 9–11: HNF1, 12–13: negative controls for HNF1, 14–15: PROS1 and 16: negative control for PROS1. The gels were run with 1X TBE buffer and supplied with 13.2 mA (100 V) for 50 min.[37]

Staining and destaining acrylamide gel

Each gel was washed with 40 ml dH2O then it was re-stained (gentle rotated by Rotatest Shaker) in 0.025% ethidium bromide solution for 15 min. After 15 min, the gel was washed with dH2O for couple of minutes and destained with 40 ml dH2O. The gel was transferred to the UV plate and the image was taken by built in software programme (XLICAP Still Image Capture V.8, 2005). [Figure 9]a and [Figure 9]b presents the results for UBC, YWHAZ, HNF1, PROS1 and their negative controls in both gels.
Figure 9: (a). Gel 1: Ladder DNA as a marker with bands size between 25 bp and 500 bp. Lane 1-3: UBC (90 bp), Lane 4-5: (−) negative controls for UBC, Lane 6-7: YWHAZ (134 bp), Lane 8: (−) negative control for YWHAZ. (b) Gel 2: Ladder DNA a as a marker and the bands size vary between 25 bp and 500 bp. Lane 9-11: hepatocyte nuclear factor 1 (122 bp) show no bands, Lane 12-13: (−) negative controls for hepatocyte nuclear factor 1, Lane 14-15: PROS1 (130 bp) show missing products. Lane 16: (−) negative control for PROS1

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  Results and Discussion Top


Despite degraded total RNA due to trapped RNA in the filter column or during prolonged centrifugation (samples), gel electrophoresis in [Figure 3] for HepG2 rRNA bands shows a good quality of RNA expression. In addition, PCR was performed to confirm that HepG2 cells express PROS1 and HNF1 as well as previous data.[38],[39],[40],[41],[42] Analysis of the total RNA quantity can be determined by spectrophotometric analysis. Dilute the RNA in TE buffer (10 mM Tris-HCl, pH 7–8, with 1 mM EDTA) and measure the absorbance at 260 and 280 nm using a quartz micro-cuvette. For best results, absorbance readings should be between 0.1 and 1.0 absorbance units. An absorbance of 1 at 260 nm corresponds to approximately 40 μg/mL of RNA. The ratio of absorbance at 260–280 nm (A260/A280) should be between 1.8 and 2.1 as suggested by the manufacturer of GenElute™ Mammalian Total RNA Miniprep Kit.

Both housekeeping genes GADPH 1 and BACTIN1 were ignored as a reference for the primers samples because they show bad amplification, non-specific binding primer dimmers as well as contamination in the control samples [Figure 9] and [Figure 10], respectively]. The best housekeeping genes [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16], [Figure 17], [Figure 18], [Figure 19], [Figure 20], [Figure 21], [Figure 22], [Figure 23], [Figure 24] were UBC [Figure 10] and YWHAZ [Figure 11]; therefore, there has been used as a reference for the primers samples in efficiency test [Figure 25], [Figure 26], [Figure 27], [Figure 28], [Figure 29]. Even though the contamination of the controls, HNF1 [Figure 12] and PROS1 [Figure 13], shows a good expression, the quality control for HNF1 [Figure 14] and PROS1 [Figure 15] and from [Figure 16] (% efficiency: for HNF1 = 36.5%; PROS1 = 53.2%) demonstrates that these primer pairs are not efficient to carry further gene knockdown experiment and required to design another HNF1 and PROS1 primers. The efficiency of the PCR should be 90%–100% meaning doubling of the amplicon at each cycle. This corresponds to a slope of –3.1 to –3.6 in the Ct vs log-template amount standard curve. To obtain accurate and reproducible results, reactions should have efficiency as close to 100% as possible (e.g., two-fold increase of amplicon at each cycle), and in any case, efficiency should be similar for both target and reference (calibrator). A number of factors can affect the efficiency of the PCR including the length of the amplicon, presence of inhibitors, secondary structure and primer design. Furthermore, the amplification efficiency must be comparable in all products and the threshold used for analysis must be within the linear phase of all the reactions, to ensure that the threshold cycle (Ct) is truly representative of initial template differences and not just a change in reaction kinetics.[43] Some studies show that gene knock down is an effective method to determine the function and effects of target genes and gene products. Previous studies [44] have proposed a role for HNF1 in regulating gene expression of PS. However, predicting that if protein HNF1 knocked down by siRNA, this could improve that protein HNF1 involved in the regulation of PROS1 gene by observing a change (decrease) in the level of PROS1 expression. To achieve this point, siRNA must be accurately designed into short products (21–23) sense from 5'-3' and antisense 3'-5' because it is quite expensive and required a good storage condition [45] that's way it is important to check the quality control of the target products before ordering the siRNA. Comparing SYBR green dye with TaqMan, the first is less sensitive because it presents a non-specific detection (primer dimers) were all dsDNA products are detected including primer dimers, contaminating DNA, and PCR product from mis-annealed primer [Figure 14], [Figure 17], [Figure 18], [Figure 19]. The specific products can be distinguished from non-specific one using melting curve (Tm) of the products. TaqMan is more accurate and has high sensitivity than SYBER green and it can be used for large quantity products. However, it not cheap (coast around £1.50 per samples) which is not preferred due to limited budget for the project.
Figure 10: Polymerase chain reaction results (melting curve)

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Figure 11: Polymerase chain reaction results (melting curve) for hepatocyte nuclear factor 1, PROS1 and five housekeeping genes: B2M1, HPRT1, RPL13A, SDHA and UBC. YWHAZ sample H2–H4 (bink); H5–H6 (green) negative controls for YWHAZ. Tm = 84

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Figure 12: Polymerase chain reaction results (melting curve) for hepatocyte nuclear factor 1, PROS1 and five housekeeping genes: B2M1, HPRT1, RPL13A, SDHA and UBC. Hepatocyte nuclear factor 1 sample A2–A4 (green); A5–A6 (pink) negative controls for hepatocyte nuclear factor 1

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Figure 13: Polymerase chain reaction results (melting curve) for hepatocyte nuclear factor 1, PROS1 and five housekeeping genes: B2M1, HPRT1, RPL13A, SDHA and UBC PROS1 sample B2–B4 (purple); B5–B6 (orange) negative controls for PROS1

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Figure 14: The 100% efficiency results (melting curves) for hepatocyte nuclear factor 1, PROS1 and two housekeeping genes: YMHAZ and UBC. B2: neat volume of hepatocyte nuclear factor 1 B3–B6: Different concentration of hepatocyte nuclear factor 1: B3: 10−1, B4: 10−2, B5: 10−3 and B6: 10−4. B7: Double distilled water as negative control; PDs: Primer dimers; melting temperature (Tm) for B2–B4 = 87;

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Figure 15: The 100% efficiency results (melting curves) for hepatocyte nuclear factor 1, PROS1 and two housekeeping genes: YMHAZ and UBC. C2: neat volume of PROS1. C3–C6: Different concentration of PROS1 C3: 10−1, C4: 10−2, C5: 10−3 and C6: 10−4. C7: Double distilled water as negative control; Tm for C2–C5 = 80; Tm for C6 = 77

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Figure 16: The 100% efficiency results (melting curves) for hepatocyte nuclear factor 1, PROS1 and two housekeeping genes: YMHAZ and UBC E2: neat volume of YMHAZ. E3–E6: UBC in different concentration: E3: 10−1, E4: 10−2, E5: 10−3 and E6: 10−4. E7: Double distilled water as negative control; Tm for E2 = 89; Tm for E3 and E4 = 80; Tm for E5 = 81; Tm for E6 = 82

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Figure 17: Polymerase chain reaction results (melting curve) for eight housekeeping genes: GADPH1, BACTIN1, B2M1, HPRT1, RPL13A, SDHA, UBC and YWHZ 1 sample A2–A4 (blue); A5–A6 (green) negative controls for GADPH 1; PDs: primer dimers

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Figure 18: Polymerase chain reaction results (melting curve) for eight housekeeping genes: GADPH1, BACTIN1, B2M1, HPRT1, RPL13A, SDHA, UBC and YWHAZ sample B2–B4 (green); B5–B6 (pink) negative controls for BACTIN1; PDs: primer dimers

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Figure 19: Polymerase chain reaction results (melting curve) for eight housekeeping genes: GADPH1, BACTIN1, B2M1, HPRT1, RPL13A, SDHA, UBC and YWHAZ sample F2–F4 (brown); F5–F6 (pink) negative controls for SDHA

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Figure 20: Polymerase chain reaction results (melting curve) for eight housekeeping genes: GADPH1, BACTIN1, B2M1, HPRT1, RPL13A, SDHA, UBC and YWHAZ B2M1 sample C2–C4 (orange); C5–C6 (dark blue) negative controls for B2M1

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Figure 21: Polymerase chain reaction results (melting curve) for eight housekeeping genes: GADPH1, BACTIN1, B2M1, HPRT1, RPL13A, SDHA, UBC and YWHAZ HPRT1 sample D2–D4 (pink); D5–D6 (green) negative controls for HPRT1

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Figure 22: Polymerase chain reaction results (melting curve) for eight housekeeping genes: GADPH1, BACTIN1, B2M1, HPRT1, RPL13A, SDHA, UBC and YWHAZ sample E2–E4 (blue); E5–E6 (pink) negative controls for RPL13A

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Figure 23: Polymerase chain reaction results (melting curve) for eight housekeeping genes: GADPH1, BACTIN1, B2M1, HPRT1, RPL13A, SDHA, UBC and YWHAZ UBC sample G2–G4 (orange); G5–G6 (green) negative controls for UBC

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Figure 24: Polymerase chain reaction results (melting curve) for eight housekeeping genes: GADPH1, BACTIN1, B2M1, HPRT1, RPL13A, SDHA, UBC and YWHAZ YWHAZ sample H2–H4 (green); H5–H6 (orange) negative controls for YWHAZ

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Figure 25: Polymerase chain reaction results (melting curve) for hepatocyte nuclear factor 1, PROS1 and five housekeeping genes: B2M1, HPRT1, RPL13A, SDHA and UBC. B2M1 sample C2–C4 (blue); C5–C6 (orange) negative controls for B2M1

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Figure 26: Polymerase chain reaction results (melting curve) for hepatocyte nuclear factor 1, PROS1 and five housekeeping genes: B2M1, HPRT1, RPL13A, SDHA and UBC. HPRT1 sample D2–D4 (blue); D5–D6 (yellow) negative controls for HPRT1

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Figure 27: Polymerase chain reaction results (melting curve) for hepatocyte nuclear factor 1, PROS1 and five housekeeping genes: B2M1, HPRT1, RPL13A, SDHA and UBC. RPL13A sample E2–E4 (red); E5–E6 (black) negative controls for RPL13A

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Figure 28: Polymerase chain reaction results (melting curve) for hepatocyte nuclear factor 1, PROS1 and five housekeeping genes: B2M1, HPRT1, RPL13A, SDHA and UBC. SDHA sample F2–F4 (blue); F5–F6 (pink) negative controls for SDHA

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Figure 29: The 100% efficiency results (melting curves) for hepatocyte nuclear factor 1, PROS1 and two housekeeping genes: YMHAZ and UBC. D2: neat volume of YWHAZ D3–D6: YMHAZ in different concentration: D3: 10−1, D4: 10−2, D5: 10−3 and D6: 10−4. D7: Double distilled water as negative control; Tm for D2 and D3 = 83; Tm for D4–D6 = 82

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Acknowledgements

Many thanks to who supported me in this study in particular my director Dr Adrian Hall, Dr Rwena Bunning and PhD students: Lindsey Bunn, Mohamed Ben-Hasan and Robert Widdowson.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16], [Figure 17], [Figure 18], [Figure 19], [Figure 20], [Figure 21], [Figure 22], [Figure 23], [Figure 24], [Figure 25], [Figure 26], [Figure 27], [Figure 28], [Figure 29]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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