發(fā)布時間：2018-04-20 00:08

  本文選題：Real-time + PCR��； 參考：《基因組學與應用生物學》2017年08期

【摘要】：分析取材前不同果糖喂養(yǎng)(禁食給清水和禁食給果糖水)條件下,大鼠肝臟常用內參基因m RNA的穩(wěn)定性,篩選出最適宜的內參,以保證實時熒光定量PCR(real-time PCR)結果的可靠性。長期給予10%果糖水建立非酒精性脂肪肝大鼠模型,在動物體重與飲用果糖量相等的情況下,取材前隨機分成兩組:禁食給清水組(Fru 1);禁食繼續(xù)給果糖水組(Fru 2);正常對照組(Con)自由飲清水。提取肝臟樣本RNA,取等量RNA逆轉錄合成c DNA,real-time PCR檢測18S、GAPDH、ACTB和TBP含量變化,分別利用ge Norm程序和Bio-Rad CFX Manager軟件分析這幾個基因的穩(wěn)定性。ge Norm分析得到的穩(wěn)定性排序為:ACTBGAPDHTBP18S。Bio-Rad CFX Manager軟件穩(wěn)定性排序為:ACTBTBPGAPDH18S。兩個軟件均得到ACTB最為穩(wěn)定,TBP次之。然而經3次重復逆轉錄基因表達分析,相對于Con組和Fru1組,Fru 2組ACTB與GAPDH明顯升高,18S略有下降,TBP在3組間比較穩(wěn)定。結合肝臟脂質合成相關的兩個關鍵基因Ch REBP,SREBP1c表達變化分析,分別以這4種基因為內參計算這兩個基因在3組間的含量變化。只有以GAPDH為內參時,Fru 2組Ch REBP表達量相對于Fru 1沒有明顯上升,其它的表達趨勢一致,只是含量高低有所區(qū)別。因此,我們認為TBP和ACTB均可作為不同果糖喂養(yǎng)狀態(tài)下大鼠肝臟的內參基因。
[Abstract]:Under the condition of different fructose feeding (fasting water and fructose water) before sampling, the stability of the commonly used internal reference gene m RNA in rat liver was analyzed, and the most suitable internal parameters were selected to ensure the reliability of the results of real-time fluorescence quantitative PCR(real-time. The rat model of non-alcoholic fatty liver was established by long-term administration of 10% fructose water. When the weight of the animal was equal to the amount of fructose consumed, Before sampling, they were randomly divided into two groups: fasting group with Fru _ (1); fasting group with fructose water group (Fru _ (2)); normal control group (Conon) with free drink of clear water. RNA was extracted from liver samples, and the content of ACTB and TBP were detected by real-time PCR of RNA reverse transcriptase synthesis. The stability of these genes was analyzed by ge Norm program and Bio-Rad CFX Manager software. Ge Norm analysis showed that the stability order of these genes was:: ACTBGAPDHTBP18S.Bio-Rad CFX Manager software:: ACTBTBPGAPDH18S. ACTB is the most stable one in both software. However, compared with Con group and Fru1 group, the expression of ACTB and GAPDH in ACTB and GAPDH increased significantly after three times repeated reverse transcriptional gene analysis. Combined with the analysis of the changes of the expression of two key genes related to liver lipid synthesis, the contents of the two genes in the three groups were calculated by using these four bases. The expression of Ch REBP in Fru 2 group was not significantly higher than that in Fru 1 group with GAPDH as the internal reference. The other expression trends were the same, but the content of Ch REBP was different from that in Fru 2 group. Therefore, we think that both TBP and ACTB can be used as internal reference genes of rat liver under different fructose feeding conditions.
【作者單位】： 重慶醫(yī)科大學基礎醫(yī)學院;中醫(yī)藥防治代謝性疾病重慶市重點實驗室;重慶醫(yī)科大學第二附屬醫(yī)院;
【基金】：國家自然科學基金(81374033,81673659) 重慶市教委課題(KJ1600233) 重慶市渝中區(qū)科委課題(20120220) 重慶醫(yī)科大學培育基金(X12100)共同資助
【分類號】：R575.5
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本文編號：1775343