《分子生物实验作业》由会员分享，可在线阅读，更多相关《分子生物实验作业（20页珍藏版）》请在金锄头文库上搜索。
1、Influence of RT-qPCR primer position on EGFR interference efficacy in lung cancer cells主题-肺癌细胞中在表皮生长因子受体干扰效能上RT-qPCR引物位置的影响。 titleAbstractvBackground:vReal-time quantitative RT-PCR (RT-qPCR) is a “gold” standard for measuring steady state mRNA levels in RNA interference assays. The knockdown of the
2、epidermal growth factor receptor (EGFR) gene with eight individual EGFR small interfering RNAs (siRNAs) was estimated by RT-qPCR using three different RT-qPCR primer sets.vResults:vOur results indicate that accurate measurement of siRNA efficacy by RT-qPCR requires careful attention for the selectio
3、n of the primers used to amplify the target EGFR mRNA.vConclusions:vWe conclude that when assessing siRNA efficacy with RT-qPCR, more than one primer set targeting different regions of the mRNA should be evaluated and at least one of these primer sets should amplify a region encompassing the siRN re
4、cognition sequence.摘要翻译v背景：实时定量逆转录聚合酶链反应技术(RT-qPCR)是在RNA干扰实验中测量稳态mRNA水平的“黄金”标准。表皮生长因子受体(EGFR)基因的敲除和八个特殊(EGFR)的小干扰RNA (siRNA)通过RT-qPCR来估测，这个RT-qPCR是三个不同的RT-qPCR引物集合。v结果：我们的研究结果表明：通过RT-qPCR的siRNA效能的精确测量要求密切注意引物的选择，引物是用来扩大EGFR mRNA.目标。v结论：我们得出的结论是当用RT-qPCR评估siRNA效能时，mRNA的不同区域上不只一个引物设置目标应当被估测并且这些引物集合中至少
5、一个应当扩大一个区域包含siRNA识别序列。BackgroundvRNA interference (RNAi) can mediate a short-term orvprolonged silencing of gene expression at the RNA andvprotein level. Knockdown efficiency is typically measuredvat the mRNA level by quantitative RT-PCR (RTqPCR)vor estimated at the protein level by immunoblot,venzy
6、me-linked immunosorbent assay (ELISA) or immunohistochemistry (IHC) 1. Many prefer measuring the relevant protein with immunoblot directly, because protein knockdown is most relevant to the observable phenotype under study.vHowever, in practice, a suitable antibody to a given target protein may not
7、always be readily available or will not allow a quantitative estimate of the magnitude of the effect of RNAi. The long turnover time of many proteins may underestimate the RNAi effect at the mRNA level. A direct measurement at the mRNA level is therefore often the preferred method to more directly v
8、erify that RNAi is effectively decreasing the amount of the transcript. Real-time RTqPCR is the “gold” standard for measuring steady-state mRNA levels. Hence, an accurate measurement method of the mRNA knockdown is needed. There are indications that mRNAs are not completely degraded after 24hrs of R
9、NAi exposure 2.vTherefore the location of the primer might be relevant as some primer sets may amplify remaining cleavage products, leading to an underestimation of the RNAi efficacy 3. Despite this,numerous publications on RNAi with RT-qPCR do not evaluate the primers choice. The lack of precise cr
10、iteria for choosing the target sequence for RT-qPCR amplification is surprising. Here, evidence is presented that the location of RT-qPCR primers is critical in the evaluation of the epidermal growth factor receptor (EGFR) small interfering RNA (siRNA) efficacy, even up to 72 hrs post-treatment in l
11、ung cancer cells.背景翻译vRNA干扰能调停短期或延长基因表达沉默在RNA和蛋白质水平上。敲除效率是在mRNA水平被典型的测量通过定量RT-PCR (RTqPCR)或在蛋白质水平被估测通过免疫印迹，酶联免疫吸附测定法(ELISA)检测或免疫组织化学方法(IHC)。很多人更喜欢直接用免疫印迹来测量相关蛋白，因为在研究中蛋白敲除是最相关的可观测的表型。然而，在现实中一个合适的给定目标的抗体不可能一直是现成的或者不允许RNA干扰影响量级的一个定量估测。很多蛋白质的覆盖时间可能低估了RNA干扰的影响在mRNA水平上。在mRNA水平上直接测量是一个非常好的方法来跟直接的证明RNA干扰是正
12、有效的减少转入的数量。实时定量逆转录聚合酶链反应技术(RT-qPCR)是对测量稳态mRNA水平的黄金标准。因此mRNA敲除的精确测量是必要的。只是mRNA不能完全的降解在RNA干扰暴露24小时后。因此引物的定位可能是与一些引物集合相关的，这些引物集可能放的保留分裂产物，导致RNA干扰效应的低估。尽管如此，在RNA干扰和RT-qPCR的众多出版物上没有评估引物的选择。对RT-qPCR扩大的选择目标序列精确标准的缺乏是令人吃惊的。在这里，呈现的证据是RT-qPCR引物的位置在表皮生长因子受体(EGFR)小干扰RNA (siRNA)效能上的评估是极其重要的，甚至在肺癌细胞上百小时的后处理。Resul
13、ts and DiscussionFigure legendvFigure 1 Determination of EGFR siRNA knockdown efficiency in H358 cells with RT-qPCR. (A) Schematic diagram of the EGFR gene exonv(boxes) structure and location of siRNAs and RT-qPCR primer sets. For the siRNAs sequences, see Table 1. For the primers sequences, seevTab
14、le 2. Primer sets were named according to the order of the sequences. (B) EGFR mRNA knockdown efficiency detected by RT-qPCRvamplification from EGFR siRNA-treated H358 cells using either the primer set q1,q2 and q3 at 48 hrs (Left), and at 72 hrs (Right). Transfectionsvwere performed in triplicate.
15、The percentages are relative to the mock treated control.图注解释v用实时定量逆转录聚合酶链反应技术在H358细胞中表皮皮生长因子受体小干扰RNA敲除效率的决定。（A)表皮生长因子受体基因外显子结构和小干扰RNA的位置还有RT-qPCR引物集。小干扰RNA序列看表1。引物序列看表2。引物集通过序列的指令来命名。（B)表皮生长因子受体MRNA敲除序列通过RT-qPCR扩增来检测，引物扩增来自于也用引物集的q1,q2 和 q3在8小时（左）和72小时（右）的表皮生长因子受体小干扰siRNA-treated H358细胞。转染是一式三份执行。百
16、分率是相关的对无效的控制。ConclusionsvWe thus conclude that there is an unexpected but significant interdependence between the EGFR targeting siRNA sequence and the RT-qPCR amplification region for assessing the efficacy of the siRNA target gene knockdown and that this finding can be extended to other mRNAs in l
17、ung cancer cells. We therefore recommend that qPCR primers for siRNA work should span the putative siRNA cleavage site, thus avoiding the amplification of mRNA molecules that underwent only the initial steps of the siRNA induced degradation process. It is also recommendable to test more than one pri
18、mer set at different locations on the mRNA, particularly for mRNA targets that appear to be refractory to siRNA-mediated cleavage. 结论翻译v结论：v这时我们得出结论：有一个不被期望但是非常重要的依赖性在表皮生长因子受体靶向小干扰RNA序列和RT-qPCR放大区域之间对评估小干扰RNA目的基因的敲除，并且这个发现在肺癌细胞中能扩展到其它mRNA。因此我们推荐：对小干扰RNA工作的qPCR引物应当跨越假定的小干扰RNA分裂位点，因此要避免mRNA分子的扩增，mRNA分
19、子是仅仅诱导降解过程的小干扰RNA的最初阶段。也推荐测试不只一个引物集在mRNA的不同位置上，尤其是mRNA目标，它的出现对小干扰RNA调节分裂是难治的。的确，引物集中至少有一个引物应当放大区域包含小干扰RNA识别序列来确保小干扰RNA有效读出的机会。MethodsvTable 1 Phenotypic consequence of EGFR downregulation by siRNAsvName ofvsiRNAvexon sequence Locationa DesignedvbyvRNA knockdown Measuredvwith q2 (%)vProteinvdownregul
20、ation (%)vViabilityv(%)vCaspase-3/v7 (%)vApoptoticvcells (%)vViable cellsv(%)vEGFRvsiRNA604v3 GCAGTCTTATCTAACTATGATGCAA C.604_628 Invitrogen 19 5 43 3 94 2 170 8 168 6 90 2vEGFRvsiRNA752v4 GCAGTGACTTTCTCAGCAA C.752_770 Eurogentec 44 13 28 1 96 2 149 6 137 6 94 1vEGFRvsiRNA1247v8-9 GCAAAGTGTGTAACGGAA
21、TAGGTAT C.1247_1271 Invitrogen 72 4 51 3 92 0 179 6 177 7 86 2vEGFRvsiRNA1608v12 GGAGATAAGTGATGGAGAT C.1608_1626 Eurogentec 25 9 11 1 97 2 132 7 131 6 94 1vEGFRvsiRNA2654v20 GGGAACACAAAGACAATAT C.2654_2672 Dharmacon 18 22 1 1 106 1 102 6 104 2 92 1vEGFRvsiRNA2708v20-v21vTCGCAAAGGGCATGAACTA C.2708_27
22、26 Dharmacon 21 9 8 1 106 1 141 6 136 6 105 2vEGFRvsiRNA3768v28 GGACTTCTTTCCCAAGGAA C.3768_3786 Eurogentec 48 8 2 1 97 2 126 6 125 6 96 1vEGFRvsiRNA4765v28 AGAATGTGGAATACCTAAGG C.4766_4785 * 2 7 2 1 108 3 146 6 137 6 93 3vNegativevsiRNAvproprietary sequence designed byvEurogentecv16 11 3 1 111 2 97
23、2 100 2 97 0vBlankvcontrolv1 5 1 0 116 2 107 4 101 1 98 1vaReference sequence identical to NM_005228.3 b Modified from 7.Figure legendvFigure 2 Down-regulation of EGFR protein levels and phenotypic consequences of EGFR knockdown. (A) Western blot was performed tovdetermine the down-regulation levels
24、 of EGFR protein. (B) Cell viability was detected by CellTiter-Blue Cell Viability Assay. (C) Caspase-3/7vactivity was measured by Apo-ONE Homogeneous Caspase-3/7 Assay. (D, E) Viable and apoptotic cells were counted with Hoechst 33342 andvpropidium iodide (PI) double fluorescent chromatin staining.
25、 The aggregate results are consistent with the mRNA knockdown results obtained invthe RT-qPCR experiments and confirm that of the siRNAs tested, the s1247 is the most powerful siRNA on H358 cells to down-regulate the EGFRvprotein level, inhibit the cell viability and induce apoptosis in comparison t
26、o other siRNAs (Results see Table 1). Idem as above.图注解释v表皮生长因子受体蛋白水平和表皮生长因子受体敲除的表型结果下调。(A)Wester印记是来执行决定表皮生长因子受体蛋白下调水平。（B）细胞活力检测是通过 CellTiter-Blue细胞活力实验。（C）Caspase-3/7活力通过Apo-ONE Homogeneous Caspase-3/7实验测试。(D, E)存活和凋亡的细胞计数通过赫斯特33342和碘化丙啶双荧光核染色质着色。在RT-qPCR实验中随着mRNA敲除结果的获得集合结果是连续的，并且小干扰RNA测试证明了这个，s1
27、247是最有能力的在H358细胞上，它来下调表皮生长因子受体蛋白水平，抑制细胞活力并且对其他小干扰RNA对照诱导细胞凋亡（结果见表1），像上面一样设计评估表vTable 2 Primer Sequences for EGFR and GAPDH Transcripts used for Real-Time Quantitative reverse TranscriptasevPolymerase Chain ReactionvName Forward exon Sequence Locationa Reverseb exon Sequence Location Lengthv(bp)vq1 E
28、GFR 370F 2 GGCACTTTTGAAGATCATTTTCTC c.370_393 EGFR 514R 3 CTGTGTTGAGGGCAATGAG c.514_532 163vq2 EGFRv1089Fv7 CGAGGGCAAATACAGCTT c.1089_1106 EGFRv1263Rv9 AAATTCACCAATACCTATT c.1263_1281 193vq3 EGFRv2034Fv15 GGCAGGAGTCATGGGAGAA c.2034_2052 EGFRv2168Rv17 GCGATGGACGGGATCTTAG c.2168_2186 153vGAPDHv240Fv3 TTGCCATCAATGACCCCTTCA c.240_260 GAPDHv395Rv5 CGCCCCACTTGATTTTGGA c.395_413 173vGAPDH GAPDHv820Fv8 TGAACGGGAAGCTCACTGG c.820_837 GAPDHv1106Rv9 TCCACCACCCTGTTGCTGTA c.1106_1125 306vGAPDHv1016Fv8 ACCCACTCCTCCACCTTTG c.1016_1034 GAPDHv1175Rv9 CTCTTGTGCTCTTGCTGGG c.1175_1193 177
