Transcriptional regulation of protein S gene

• Main Authors: Maha Dawood Jaffarali, Adrian Hall
• Format: Article
• Language: English
• Published: Wolters Kluwer Medknow Publications 2019-01-01
• Series: Hamdan Medical Journal
• Subjects: protein s; hnf1; housekeeping genes
• Online Access: http://www.hamdanjournal.org/article.asp?issn=2227-2437;year=2019;volume=12;issue=4;spage=170;epage=181;aulast=Jaffarali
• ISSN: 2227-2437; 2227-247X

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.