SymbolConstruction of forkhead transcription factor 1 plasmid standard

BACKGROUND:

Studies show that forkhead transcription factor 1 (Fox01) plays an important role in insulin resistance and the pathogenesis of type 2 diabetes. However, research regarding diversity of normal Fox01 and diabetics are few. Therefore, it is necessary to construct the plasmid standard.

OBJECTIVE:

To construct the plasmids standard of fox01mRNA, in addition, to detect the standard by the method of RT-PCR.

DESIGN, TIME AND SETTING:

The experiment of repeating single sample was performed at the First Affiliated Hospital of Soochow University Laboratory from November 2007 to March 2008.

MATERIALS:

SYBR ExScriptTMRT-PCR Kit, SYBR Premix Ex TaqTM kits, TaKaRa agarose gel DNA purification kit, TaKaRa Minibest plasmid DNA purification kit, PMD18-T Vector Kit, and other reagents, primers and equipments.

METHODS:

The total RNA was extracted from the blood samples of healthy volunteers. Then cDNA was synthesized by reverse transcription. The target sequence was amplified from cDNA, and then the PCR product was gel extracted and purified. Products of purification were connected to PMD18-T vector and transformed into competent cells JM109. The method of sequencing was used to verify that the target fragments were transformed successfully. Plasmid DNA was extracted and was measured using UV spectrophotometer and converted to copies/mL according the equation. Finally it was diluted into a standard gradient.

MAIN OUTCOME MEASURES:

The results of recombinant plasmid PCR and gene sequencing, the correlation coefficient of the standard curve and the repeatability of the plasmid standard.

RESULTS:

A plasmid standard for Fox01 was successfully constructed. The methods of sequencing and PCR verified that the target fragments were transformed successfully. After amplified by FQ-PCR, the standards of the different concentration had good linear relationship between the cycle threshold and the numerical of the start template. The linear range was 2.39× (10⁵-10¹²) copies/mL. The standards of the different concentration had good repeatability, and their variation coefficient was in the range of 0.29%-1.16%.

CONCLUSION:

Fox01 plasmid standard, which has a better sensitivity and repeatability, is successfully constructed.

Cai HF, Shi JF, Gu GH. Construction of forkhead transcription factor 1 plasmid standard.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2009;13(7): 1263–1266(China) [http://www.crter.cnhttp://en.zglckf.com]