Data supporting the design and evaluation of a universal primer pair for pseudogene-free amplification of HPRT1 in real-time PCR

نویسندگان

  • Reza Valadan
  • Akbar Hedayatizadeh-Omran
  • Mahdyieh Naghavi Alhosseini-Abyazani
  • Omolbanin Amjadi
  • Alireza Rafiei
  • Mohsen Tehrani
  • Reza Alizadeh-Navaei
چکیده

Hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) is a common housekeeping gene for sample normalization in the quantitative reverse transcriptase polymerase chain (qRT-PCR). However, co-amplification of HPRT1 pseudogenes may affect accurate results obtained in qRT-PCR. We designed a primer pair (HPSF) for pseudogene-free amplification of HPRT1 in qRT-PCR [1]. We showed specific amplification of HPRT1 mRNA in some common laboratory cell lines, including HeLa, NIH/3T3, CHO, BHK, COS-7 and VERO. This article provides data supporting the presence and location of HPRT1 pseudogenes within human and mouse genome, and the strategies used for designing primers that avoid the co-amplification of contaminating pseudogenes in qRT-PCR. In silico analysis of human genome showed three homologous sequences for HPRT1 on chromosomes 4, 5 and 11. The mRNA sequence of HPRT1 was aligned with the pseudogenes, and the primers were designed toward 5' end of HPRT1 mRNA that was only specific to HPRT1 mRNA not to the pseudogenes. The standard curve plot generated by HPSF primers showed the correlation coefficient of 0.999 and the reaction efficiency of 99.5%. Our findings suggest that HPSF primers can be recommended as a candidate primer pair for accurate and reproducible qRT-PCR assays.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

ارزیابی روش Real-time PCR Multiplex به‌منظور شناسایی گونه‌های شایع باکتری بروسلا

Background and purpose: Brucellosis is a zoonotic disease. Given that serologic test has some specific characteristics such as difficult growth, problems associated with culture, and cross-reactions with otherbacteria, in this study multiplex real-time polymerase chain reaction (PCR) method was used to detect the most common species of Brucella spp. Materials and methods: This experimental s...

متن کامل

A focused Real Time PCR strategy to determine GILZ expression in mouse tissues

Glucocorticoid-Induced Leucine Zipper (GILZ) is a glucocorticoid-inducible gene that mediates glucocorticoid anti-inflammatory effects. GILZ and the isoform L-GILZ are expressed in a variety of cell types, especially of hematopoietic origin, including macrophages, lymphocytes and epithelial cells, and strongly upregulated upon glucocorticoid treatment. A quantitative analysis of GILZ expression...

متن کامل

Rapid Detection of COVID-19 by RT-LAMP PCR Technique and its Comparison with Real-Time RT-PCR Method

Rapid antigen and antibody, serological tests, and RT ‑ PCR-based molecular methods are widely used for the detection of microorganisms worldwide. Objectives: This study aimed by Isothermal nucleic acid amplification techniques to detect the covid-19. Methods: In this study, 200 samples of nasopharynx and oropharynx were collected from Jamaran Heart Hospital in Tehran. Covid -19 was exami...

متن کامل

Analysis of Herpes Simplex Virus in Suspected Encephalitis, Keratitis and Dermal Infections Using Real- Time PCR

Background & Objectives: Herpes viruses can cause diseases in the clinical range. The virus can cause infection in various body parts, especially eyes and nervous system. The aim of this study was at evaluating  the Real-Time TaqMan probe PCR in diagnosing and monitoring of the patients with suspected HSV infections.Materials & Methods: More than a thousand patients with suspected HSV infection...

متن کامل

Designing Of Degenerate Primers-Based Polymerase Chain Reaction (PCR) For Amplification Of WD40 Repeat-Containing Proteins Using Local Allignment Search Method

Degenerate primers-based polymerase chain reaction (PCR) are commonly used for isolation of unidentified gene sequences in related organisms. For designing the degenerate primers, we propose the use of local alignment search method for searching the conserved regions long enough to design an acceptable primer pair. To test this method, a WD40 repeat-containing domain protein from Beauveria bass...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:

دوره 4  شماره 

صفحات  -

تاریخ انتشار 2015