Question

半定量RT-PCR内标怎样选择

Answer 1

搜集了几个植物RT-PCR的内标,与各位共享
1． 物种：拟南芥（arabidopsis）或maize
2． 基因名称：18S rRNA
3． 模板：cDNA
4． PCR类型及目的：RT-PCR
5． Forward primer (5’-3’): CCATAAACGATGCCGGA
Reverse primer (5’-3’): CACCACCCATAGAATCAAGA
Probe: 无
6． 产物长度（bp）：350
7． 引物浓度：50 pmol/ul
8． 退火温度：54.1
9． 所用仪器：PE-9600
10． 提交者ID：yuxing955
11． 提交者联系方式：xing-yu955@163.com
12． 参考文献原文：本人亲自验证，还有本实验室其他人亲自验证。

1． 物种： maize
2． 基因名称：actin
3． 模板：cDNA
4． PCR类型及目的：RT-PCR
5． Forward primer (5’-3’): AAATGACGCAGATTATGTTTGA
Reverse primer (5’-3’): GCTCGTAGTGAGGGAGTACC
Probe: 无
6． 产物长度（bp）：
7． 引物浓度：50 pmol/ul
8． 退火温度：54.1
9． 所用仪器：PE-9600
10． 提交者ID：yuxing955
11． 提交者联系方式：xing-yu955@163.com
12． 参考文献原文：本人亲自验证，还有本实验室其他人亲自验证

Semiquantitative RT-PCR
Reverse transcriptions were carried out using the Super Script II RT and the adapter primer of the 3' RACE System. For amplification of ROT4 cDNA, ROT4-F and ROT4-R were used. As a control, the following oligonucleotides were used to detect the constitutively expressed ACTIN2 (ACT2) gene (An et al., 1996): ACT2-F, 5'-GAAATCACAGCACTTGCACC-3'; ACT2-R, 5'-AAGCCTTTGATCTTGAGAGC-3'. The condition for amplification by RT-PCR was one cycle at 50°C for 15 min and 94°C for 2 min, then 27 cycles for ROT4 cDNA amplification or 33 cycles for ACT2 cDNA amplification at 94°C for 15 sec, 57°C for 30 sec, and 72°C for 60 sec.

可以
The Arabidopsis AtACT8 (actin 8) gene was used as a positive internal control (An et al. 1996). The PCR primers used to detect AtACT8 mRNA were 5'-ATG AAG ATT AAG GTC GTG GC-3' and 5'-TCC GAG TTT GAA GAG GCT AC-3' (Aida et al. 1997). These products were fractionated by electrophoresis on a 3% agarose gel and stained with ethidium bromide, and scanned using a fluorescent image analyser.
。

UBQ, 5_-GATCTTTGCCGGAAAACAATTGGAGGATGGT-3_
and 5_-CGACTTGTCATTAGAAAGAAAGAGATAACAGG-3_.

Quantitative PCR Analysis of GSL mRNA
Total RNA (5 μg) was used in cDNA reactions using the Superscript II cDNA synthesis kit (Invitrogen). The cDNA was diluted 2.5-fold, and 1 μL was taken for quantitative real-time PCR in 20-μL reaction volumes using 10 μL of 2× Quanti-Tect PCR master mix (Qiagen, Valencia, CA), 0.3 μM gene-specific primers, and 0.6 μL of a 100-fold dilution of SYBR Green I dye (Applied Biosystems, Foster City, CA). PCR cycling and fluorescence measurements were performed with a Rotorgene 2000 Real-Time Cycler RG2072 (Corbett, Sydney, Australia), and data were normalized against glyceraldehyde phosphate dehydrogenase (GAPDH), actin (Actin1), and cyclophilin (Cyclo) mRNA levels (Vandesompele et al., 2002 ). Primers used in the quantitative PCR were as follows: for AtGAPDH (At3g26650), 5′-TGGTTGATCTCGTTGTGCAGGTCTC-3′/5′-GTCAGCCAAGTCAACAACTCTCTG-3′; for AtActin1 (At2g37620), 5′-TGCGACAATGGAACTGGAATG-3′/5′-GGATAGCATGTGGAAGTGCATAC-3′; for AtCyclo (At2g36130), 5′-TGGCGAACGCTGGTCCTAATACA-3′/5′-CAAAAACTCCTCTGCCCCAATCAA-3′; for AtGSL5 (At4g03550), 5′-CTGGAATGCTGTTGTCTCTGTTG-3′/5′-TCGCCTTTTGATTTCTTCCCAGT-3′; for AtGSL6 (At1g05570), 5′-GAAGGGTTTGGGCGTTGGAAG-3′/5′-CAATGAGAAGCATTCCCCATCCAGTT-3′; and for AtGSL11 (At3g59100), 5′-TTTAGGGGTTTGGGACTCGGTGAAA-3′/5′-TGTCTTTCCGACCAGCGAGAATCA-3′.