实验目的：学习RT-PCR的原理及其操作过程。
实验材料：水稻叶片的RNA。
实验原理：目前PCR技术只能扩增DNA模板，对 RNA模板不能直接扩增。mRNA 反转录生成的cDNA可作为PCR的模板进行扩增，这种在mRNA反转录后进行的PCR扩增称为RT-PCR。RT- PCR比Northern杂交更灵敏，对RNA的质量要求较低，操作简便，它是在转录水平上检测基因时空表达的常用方法。
本实验以水稻叶片 RNA为材料，检测β-actin基因的表达。实验中设定2个阴性对照：一个不加模板RNA，另一个不加反转录酶，主要是消除DNA及PCR试剂方面引起的假阳性；同时以叶片DNA为阳性对照，检验PCR试剂和扩增过程是否有问题。
实验步骤：
RNA Preparation
4. Incubate at 37℃ for 15 min, then 70℃ for 10 min.
Reverse Transcriptase Reaction
1. Add 1 ml 500 mg / ml oligo (dT) 15 primer, mix the contents of the tube by gently vortexing and collect the reaction by brief centrifugation.
2. Heat the mixture at 65℃ dry bath for 10 minutes, then place at room temperature for 10 minutes. Add the following contents:
5 × first strand buffer 4 ml
0.1 M DTT 2 ml
10 mM dNTP 1 ml
3. Mix the contents of the tube by gently vortexing and collect the reaction by brief centrifugation. Place the tube at 37℃ water bath for 2 min.
4. Add 2 ml 200 U / ml M-MLV Reverse Transcriptase. Mix gently and incubate at 37℃ for 1 h. The total volume should now be 20 ml.
5. Inactive the reaction by heating at 70℃ for 15 min, then add 20 ml ddH2O and the first strand of cDNA can be used as a template to amplification in PCR.
6. To remove the RNA complementary to the cDNA, add 1 ml (2 units) of RNase H and incubate at 37℃ for 20 min.
PCR Amplification
1. Prepare the mixture containing the following on ice:
cDNA first strand 1 ml
TaKaRa Ex Taq (5 u / 1 ml) 0.5 ml
10 × Ex Taq buffer 5 ml
dNTP mixture (2 mM) 4 ml
Primer F (10 mM) 2 ml
Primer R (10 mM) 2 ml
ddH2O 35.5 ml
2. Commence PCR program
94℃ 60℃ 72℃ 4℃ Cycles
Step 1 3 min 1
Step 2 1min 1 min 1 min 30-40
Step 3 5 min 1
Step 4 forever
3. The β-actin is used as the internal standard for each RT-PCR, DNA contamination in the RNA sample is tested by replacing the reverse transcriptase with water in the RT-PCR.
Reagents
5 × first strand buffer (GIBCO Part No.Y00146)
100 mM DTT (GIBCO Part No.Y11147)
200 U / ml M-MLV Reverse transcriptase (GIBCO Part No.28025-021)
500 mg / ml oligo (dT) 15 primer (Promega Cat. No. C110A 9362612)
10 mM dNTP mix
RNase-free DNase I (TaKaRa Code No. 2215 CA)
5 U / ml Ex Taq polymerase and 10 × buffer (TaKaRa Code No. RR001D CA)
10 ml M gene specific Primer F and R
Sterile ddH2O
Mineral oil
