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RT-PCR从实验到写作,零基础教程

PCR,相信我们都不陌生,是一种选择性体外扩增DNA或RNA片段的方法,即通过试管中进行的DNA复制反应,使极少量的基因组DNA或RNA样品中特定基因片段在短短几小时内扩增上百万倍。

简单说,就是我们在体外模仿体内DNA复制的过程,混合DNA模板、合适的引物、足够的4种dNTP、耐热DNA聚合酶以及适宜的体系后,在一定温度和时间条件下,进行的DNA扩增。

PCR有很多种,其中RT-PCR,全称反转录聚合酶链反应(reverse transcription-polymerase chain reaction, RT-PCR)是将RNA的反转录和cDNA的聚合酶链式扩增(PCR)相结合的技术。

实验原理是在试管内,以变性-退火-延伸三个基本反应为一个循环,反复重复这种循环,使DNA得以扩增。其目的是检测核酸的表达,主要是RNA的表达,DNA不需要反转录的过程,直接PCR检测即可。

一、RT-PCR操作步骤

1. RNA抽提

方法:TRlzol法。

主要成分:苯酚(具有裂解细胞和变性蛋白的作用)。

RNA抽提前准备:试剂耗材准备(注意RNase free)、样品的准备。

步骤:样品+1ml TRlzol室温裂解10min+200μL氯仿振荡30s后室温静置2-3 min;4℃低温离心(12 000g×20min);400μL上层水相+600μL异丙醇室温沉淀10min;4℃低温离心(12 000g×20min);75%乙醇冲洗;晾干沉淀,DEPC-treated水溶解。

2. RNA 质量检测

方法:分光光度计测定、凝胶电泳法

3. 反转录

是以RNA为模板合成cDNA的过程。在这个过程中遗传信息的流动方向和转录时相反,所以称为“反转录”。在这个过程中需要一个反转录酶,合成的DNA称为与RNA互补的DNA(complementary DNA, cDNA)。这个过程中根据不同的情况选择不同的反转录引物,如果要检测的RNA有poly A尾巴,可以用Oligo(dT)做反转录引物;如果要检测的RNA没有poly A尾巴,就用随机引物(random primer)来反转;如果要检测的RNA序列已知,可用特定引物(specific primer)来反转。

4. PCR程序

三步法:

第一步:94℃变性30s;

第二步(循环30-35次):94℃变性30s,55-60℃退火30s,72℃延伸24s(时间按照合成速度和长度来计算);

第三步:72℃终止延伸5-10min。

5. 引物

引物设计步骤:

找到目的基因的CDs序列(存在多个转录本时可对各个转录本的保守区域进行引物设计);用软件设计和评价引物(Oligo软件);blast引物特异性。

要求:

引物长度为15-30bp,最常用的为23bp;引物Tm值介于62-73℃之间,上下游引物Tm最好相近以保证其能高效工作;3’-端的Tm值要低于5’端;引物GC含量约为40-60%;避免扩增模板的二级结构域;避免引物的二级结构;避免与靶DNA错配。

6. PCR酶:可按需要选择普通的Taq酶、可扩增长片段的Taq酶或高保真酶。

7. PCR体系:按所选的PCR酶说明书来配制。

8. PCR产物检测:一般采用琼脂糖凝胶电泳,必要时也可使用聚丙烯酰胺凝胶电泳。

反转录引物选择:

1. Oligo dT:适用于具有PolyA尾巴的RNA。(原核生物的RNA、真核生物的rRNA和tRNA不具有PolyA尾巴。)由于Oligo dT要结合到PolyA尾巴上,所以对RNA样品的质量要求较高,即使有少量降解也会使全长cDNA合成量大大减少。

2. 随机引物(random primer): 适用于长的或具有发卡结构的RNA。适用于rRNA、mRNA、tRNA 等所有RNA的反转录反应。

3. 特异性引物(Specific primer):与模板序列互补的引物,适用于目的序列已知的情况。

二、RT-PCR需注意的细节

1. 实验过程中必须戴口罩、手套,尽量避免其他人员干扰。

2. RT试剂盒从冰箱拿出,应该充分解冻,瞬离后,混匀,放于冰上,可在冰上加样。试剂可以用数字贴纸编号,避免漏加、错加试剂。

3. 取用不同的试剂时,必须更换枪头,样本之间要避免交叉污染。

4. 逆转录时,PCR仪可以先预热。

5. 反应体系配好之后,应充分混匀,瞬离,并且弹去管内的气泡。

6. 实验过程中应预防RNA酶污染,使用无酶枪头以及EP管,同时台面可以使用固相RNA酶去除剂清洁。

7. PCR时,管壁尽量避免写字。

8. 管盖一定要盖严。

9. 10μL逆转录反应体系建议加入不超过500ng的RNA。

10. 如果目的基因表达量非常低,最多加入1μg Total RNA,否则加入RNA量过高,可能会超出后续PCR的线性范围。

11. 反应体积可根据需要等比例放大。

12. 试剂尽量不要反复冻融。

13. 每个样品至少3个平行孔,所有成分加完后,离心去除气泡。

14. 可将所有共同物质加入同一管中,混匀后分散入其他管中。

三、RT-PCR写作例句

第一句

Total RNA was extracted with/ isolated from cultured cells/ samples/ tumor tissues using/ by Trizol reagent (Invitrogen, Thermo Fisher Scientific, Braunschweig, Germany)/ RNeasy kit (Qiagen)/ RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol /following the manufacturer’s (recommended) instructions as described previously.
TRIzol reagent (Thermo Fisher Scientific) was used to extract RNA from samples.

第二句(可与第一句合并)

RNA (2 μg) was converted into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo).
Total RNA was reverse-transcribed into cDNA using the aScript cDNA SuperMix (Quanta Biosciences).
2 μg RNA was used to synthesize cDNA using the PrimeScript RTreagent kit (Takara, DRR037A).

第三句

Gene primers (…) were designed using Primer Express v3.0 Software.
The primer sequences used are listed/shown in Supplemental Experimental Procedures/ Table X. 也可写与句末。
The following primers were used:/ Gene-specific primers as follows: Gene名称forward 5′-XXX-3′ and reverse 5′- XXX-3′;

第四句

RT-PCR was performed with/ accomplished using/ carried out using …according to the manufacturer’s instructions.

第五句

GADPH /U6 was used as endogenous controls/ an internal reference. Relative expression level was computed using 2-ΔΔCt method.
For the data of each sample, the Ct value of the target gene was normalized to that of U6 (…).
The expression level of each gene was performed and the data were normalized to control unwanted sources of variation.
The relative expression level of indicated genes was compared with that of… and expression fold changes were calculated using 2-△△Ct methods.

第六句(可写可不写)

Each qRT-PCR reaction was performed in triplicate as follows: step 1: denaturation at 95°C for 10 min; step 2: 40 cycles of 95°C for 15 s and 60°C for 1 min.
PCRs were a relative estimation in triplicate as per the following temperature profile: denaturation 95 °C for 10 min followed by amplification by 40 cycles of 95 °C for 10 s and 60 °C for 1 min.

第七句(可写可不写)The cDNA was then diluted to 70 μL in water. qPCR was performed with 2×SensiFAST SYBR No-ROX kit (Bioline), gene-specific primers (250 nM final concentration of forward and reverse), and 2 μL cDNA.

举例

1. Total RNA was extracted using a Trizol reagent (Invitrogen). RNA (2 μg) was converted into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo). QRT-PCR was accomplished using the FastStart Universal SYBR Green Master (Rox) (Roche) in the ABI PRISM® 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA). GADPH and U6 were used as endogenous controls. We used melting curves to monitor non-specific amplifications. Relative expression level was computed using 2-ΔΔCt method. The primer sequences used are listed in Supplemental Experimental Procedures.

2. Total RNA was extracted by TRIzol reagent as described previously xx. The expression levels of 7 randomly selected differentially expressing circRNAs (Fold changes ≥ 2, p < 0.05) were measured by qRT-PCR; among them, 2 were upregulated and 5 were downregulated in the GC tissues: (upregulated: hsa_circ_0081146, hsa_circ_0084720), (downregulated: hsa_circ_0060108, hsa_circ_0057104, hsa_circ_0054971, hsa_circ_0063561, and hsa_circ_0058766). GAPDH expression was used as an internal reference. The primers used for these amplifications are listed in Table S1. PCRs were a relative estimation in triplicate as per the following temperature profile: denaturation 95 °C for 10 min followed by amplification by 40 cycles of 95 °C for 10 s and 60 °C for 1 min.

3. Total mRNA was isolated using Trizol (Invitrogen) and 2 μg RNA was used to synthesize cDNA using the PrimeScript RTreagent kit (Takara, DRR037A) according to the manufacturer’s protocol. Real-time PCR was performed using FastStart Universal Probe Master Mix (Roche) and analyzed with a Stratagene Mx 3000P thermal cycler. Real-Time PCR primer sequences are listed in Supplementary information, Table S1.

4. Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions, reverse transcribed and subjected to real-time PCR as previously described. [XX] Expression data were normalized to the geometric mean of the housekeeping gene GAPDH to control the variability in expression levels and calculated as 2- [(CT of indicated genes) – (CT of GAPDH)], where CT represents the threshold cycle for each transcript.

5. The total miRNA samples were extracted from ASPC-1 cells, PANC-1 cells and tumor tissues using SanPrep Column microRNA Mini-Preps Kit (Sangon Biological Engineering Technology & Services Co., Ltd., China) following the manufacturer’s protocol. The purity of the extracted miRNA was determined, and the RT-PCR was carried out using a microRNA First Strand cDNA Synthesis Kit following the manufacturer’s instructions with a TC-512 PCR system (TECHNE, UK). Quantitative real-time PCR assay was performed with a MicroRNAs Quantitation PCR Kit (Sangon Biological Engineering Tech- nology & Services Co., Ltd., China) and ABI 7500 Real Time PCR System (Applied Biosystems, USA). For the data of each sample, the Ct value of the target gene was normalized to that of U6 (Sangon Biological Engineering Technology & Services Co., Ltd., China). The unknown template was calculated using the standard curve for quantitative analysis. Eventually, the expression level of each gene was performed and the data were normalized to control unwanted sources of variation.

6. The total RNA of the tissue samples was extracted using the Trizol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was converted from total RNA by using a Reverse Transcription Kit (Takara, Dalian, China) according to the manufacturer’s instructions. Quantitative real-time PCR was performed with SYBR Green (Takara, Dalian, China),0 and the data collection was performed on the Applied Biosystems® 7500 Real-Time PCR Systems (Thermo Fisher Scientific) according to the manufacturer’s instructions. The primers were synthesized by Biosune (Shanghai, China). The relative expression level of indicated genes was compared with that of β-actin and expression fold changes were calculated using 2-△△Ct methods. Each qRT-PCR reaction was performed in triplicate. Sequences of primers used for qRT-PCR in this study are shown in Additional file 1: Table S2.

7. Total RNA was extracted from cell lines, using Trizol total RNA isolation reagent (Invitrogen), according to the manufacturer’s specifications and treated with Turbo DNase (Ambion). cDNA was synthesized from total RNA (0.5 mg) using random hexamers with TaqMan cDNA Reverse Transcription Kit (Applied Biosystems). Gene primers (Gabra3 F-5’- GACCACGCCCAACAAGCT-3’; R-5’-AGCATGAATTGTTAACCTCATTGTATAGA-3’; GAPDH-F-5’-GAAGGTGAAGGTCGGAGTCAAC; R-5’-CAGAGT- TAAAAGCAGCCCTGGT-3’) were designed using Primer Express v3.0 Software and real-time PCR was performed using SYBR Select Master Mix (Applied Bio- systems). All reactions were carried out on the 7500 Fast Real-Time PCR System (Applied Biosystem). The average of three independent analyses for each gene and sample was calculated using the DD threshold cycle (Ct) method and was normalized to the endogenous reference control gene GAPDH.

8. TRIzol reagent (Thermo Fisher Scientific) was used to extract RNA from samples. Then a total of 250 ng of RNA was used to perform reverse transcription with QuantiMir RT kit (System Biosciences, Palo Alto, CA, USA). The primers used were as follows: CISD2, 5’-GCAAGGTAGCCAAGAAGTGC-3’ (forward) and 5’-CCCAGTCCCTGAAAGCATTA-3’ (reverse); GAPDH, 5’-GCGAGATCGCACTCATCATCT-3’ (forward) and 5’-TCAGTGGTGGACCTGACC-3’ (reverse). CISD2 was amplified as follows: step 1: denaturation at 95°C for 10 min; step 2: 40 cycles of 95°C for 15 s and 60°C for 1 min. Results were calculated with 2−DDCT and normalized to GAPDH.

9. Total RNA was extracted from cultured MCF7, A549 and UACC903 cells using Trizol reagent (Takara, Shiga, Japan) according to the manufacturer’s instruction. Reverse transcription was carried out with 0.5 μg total RNA using the PrimerScript™ RT reagent kit (Takara, Shiga, Japan). After incubation for 1 h at 42 °C, the reactions were terminated by heating at 70 °C for 15 min. To analyze alternative splicing of exon 2 in the Bcl-x gene, 5′ primer to Bcl-x (5′-GAGGCAGGCGACGAGTTTGAA-3′) and 3′ primer (5′-TGGGAGGGTAGAGTGGATGGT-3′) were used for PCR amplification (30 cycles, 94 °C, 30s; 56 °C, 30s; 72 °C, 1 min) with 2xEasy Taq superMix (Transgen Biotech, China). The length of splicing variants of Bcl-xL and Bcl-xS are 460 bp and 271 bp respectively. To analyze alternative splicing of exon 3, 4, 5, 6 in the Caspase 9 gene, 5′ primer to Caspase 9 (5′-GCTCTTCCTTTGTTCATCTCC-3′) and 3′ primer (5′-CATCTGGCTCGGGGTTACTGC-3′) were used for PCR amplification (30 cycles, 94 °C, 30s; 54 °C, 30s; 72 °C, 1 min). The length of splicing variants of Caspase 9a and Caspase 9b are 742 bp and 292 bp, respectively. PCR products were separated and analyzed on agarose gels, with the bands of the Bcl-x and Caspase 9 splicing variants being confirmed by DNA sequencing.

10. Brain tissues from the ipsilateral SN were dissected at the indicated time points after LPS or PBS injection with capsaicin or vehicle, and total RNA was extracted in a single step using RNAzol B (Tel-Test, Friendswood, TX) following the instructions of the manufacturer. Total RNA was reverse transcribed into cDNA using AMV reverse transcriptase (Promega, Madison, WI) and random primers (Promega). The primer sequences used in this study were as follows: 5′-TGATGTTCCCATTAGACAGC-3′ (forward) and 5′-GAGGTGCTGATGTACCAG TT-3′ (reverse) for IL-1β (378 bp); 5′-ACACTCTATCACTGGCATCC-3′ (forward) and 5′-AAGGGACACCCTTTCACAT-3′ (reverse) for COX-2; 5′-GCAGAA TGTGACCATCATGG-3′ (forward) and 5′-ACAACCTTGGTGTTGAAGGC-3′ (reverse) for iNOS (557 bp); and 5′-TCCCTCAAGATTGTCAGCAA-3′ (forward) and 5′-AGATCCACAACGGATACATT-3′ (reverse) for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 308 bp). The PCR amplification consisted of 30 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s (for IL-1β and GAPDH or 54 °C for 30 s for iNOS) and extension at 72 °C for 90 s. PCR products were separated by electrophoresis on 1.5% agarose gels, after which the gels were stained with ethidium bromide and photographed. For semiquantitative analyses, the photographs were scanned using a Computer Imaging Device and the accompanying software (Fujifilm, Tokyo, Japan).

作者: 解螺旋

解螺旋-中国临床医生的科研成长平台。医生做科研就像减肥,目的是让自己变得更好,但过程却无比痛苦。我们通过解构科研规律、归纳文章套路,授医生以渔。用最优的效率,令你成为一位懂科研的临床医生。搜索微信公众号HelixLife,关注我们吧



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