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Messenger RNA expression analysis using quantitative real-time PCR Many mRNA and primer sequences have already been identified in pigs [20-22]. When genes were not described in this species, tBLASTn searches of the GenBank and PEDEblast ESTs databases, using known human and murine amino acid sequences, have been performed.These primers (purchased from Eurogentec) allowed the mRNA expression analysis of various genes involved in the innate immune response (Table 1). The qPCR was performed using cDNA synthesized as previously described [23]. Diluted cDNA (10X) was combined with primer/probe sets and IQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s recommendations. The qPCR conditions were 98°C for 30 s, followed by 37 cycles with denaturation at 95°C for 15 s and annealing/elongation for 30 s (annealing temperature,Table 1). Real time assays were run on a Bio-Rad Chromo4 (Bio-Rad). The specificity of the qPCR reactions was assessed by analyzing the melting curves of the products and size verification of the amplicons. Each qPCR reaction included a reverse transcription negative control (RNA sample without reverse transcriptase) to check the absence of genomic DNA. To minimize sample variation, we used identical number of cells and high quality RNA. Samples were normalized internally using simultaneously the average cycle threshold (Cq) of Hypoxanthine PhosphoRibosyl-Transferase 1 (HPRT-1), Ribosomal Protein L 19 (RPL-19) and Tata Box Binding Protein 1 (TBP-1) [24] as references in each sample to avoid any artifact of variation in the target gene. These genes were selected as the reference genes because of their low variation between samples. A standard curve was generated using diluted cDNA. The correlation coefficients of the standard curves were > 0.995 and the concentrations of the test samples were calculated from the standard curves, according to the formula y = -M × Cq +B, where M is the slope of the curve, Cq the point during the exponential phase of amplification in which the fluorescent signal is first recorded as being statistically significant above background and B the y-axis intercept. Cq values were used to calculate the qPCR efficiency from the given slope according to the equation: qPCR efficiency = (10[-1/M] - 1) × 100. All qPCRs displayed efficiency between 90% and 110%. Expression data are expressed as relative values after Genex macro-analysis with three reference genes (Bio-Rad, Hercules, USA) [25]. |
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sunrq516516
金蟲 (小有名氣)
- 翻譯EPI: 7
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【答案】應助回帖
★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★
愛與雨下: 金幣+2 2014-04-07 14:43:23
868788: 金幣+30, 翻譯EPI+1, ★★★很有幫助 2014-04-07 15:11:03
愛與雨下: 金幣+2 2014-04-07 14:43:23
868788: 金幣+30, 翻譯EPI+1, ★★★很有幫助 2014-04-07 15:11:03
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許多mRNA和引物序列已經(jīng)在豬中被鑒定出來。當在無法在豬這個物種中找到（目標）基因時，tBLASTn利用已知的人類和鼠類的氨基酸序列查找了基因Bank和PEDEblast ESTs數(shù)據(jù)庫。這些引物（從eruo...買的）可以分析多種參與先天免疫應答的基因的mRNA表達（表1）跟之前描述的一樣（看文獻23），qPCR是通過cDNA合成來實現(xiàn)的（呃，就是先合成cDNA，再PCR）。稀釋后的cDNA（10倍）根據(jù)使用手冊上建議的方法與引物/探針組以及SYBR Green(XXX)結(jié)合。qPCR的條件是98度30秒，接著是在37個循環(huán)（95度變性15秒-低溫退火/延伸30秒（溫度見表1））。實時分析是用bio-rad chromo4這個儀器來跑的。qPCR反應的特異性是通過產(chǎn)物的溶解曲線以及擴增子的大小來評估的。每個qPCR反應包括一個反轉(zhuǎn)錄陰性對照（RNA樣品，不要放反轉(zhuǎn)錄酶），這是用來確認樣品里沒有DNA的。為了使樣品的變動最小化，我們使用了等量的細胞數(shù)和高質(zhì)量的RNA。樣品是采用內(nèi)標法來計算的，這里同時使用了HPRT-1，RPL-19和TBP-1的Cq作為內(nèi)標（參考文獻24），這樣做是為了避免由對單個樣品處理時人工操作而引起的對目標基因的誤差。選擇這些基因（作為內(nèi)標）是因為文獻中說它們在不同的樣品中變化都比較小。標準曲線是根據(jù)cDNA的（梯度）稀釋算出來的。標準曲線的相關(guān)系數(shù)大于0.995。待測樣品的濃度是根據(jù)標準曲線來計算的，公式是y = -M × Cq +B。其中M是曲線仙侶，cq是在擴增的指數(shù)階段，當熒光信號和背景產(chǎn)生顯著統(tǒng)計差異時被記錄下來的第一個點，B則是和Y軸的截距。cq值是根據(jù)已知的斜率用來計算qPCR的效率。等式是：qPCR efficiency= (10[-1/M] - 1) × 100.所有的qPCR的效率大約在90%到110%之間。用GeneX macro-analysis分析得到的基因表達結(jié)果是和三個內(nèi)標基因比較的相對值。 呃……that's it.... |
2樓2014-04-07 14:41:29
sunrq516516
金蟲 (小有名氣)
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- 注冊: 2013-10-17
- 性別: MM
- 專業(yè): 代謝性疾病藥物藥理
3樓2014-04-07 14:51:46
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