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. 2007 May 28;13(20):2883-8.
doi: 10.3748/wjg.v13.i20.2883.

Detection of let-7a microRNA by real-time PCR in gastric carcinoma

Hong-He Zhang  1 , Xian-Jun Wang, Guo-Xiong Li, En Yang, Ning-Min Yang
Affiliations

Detection of let-7a microRNA by real-time PCR in gastric carcinoma

Hong-He Zhang et al. World J Gastroenterol. .

Abstract

Aim: To establish an accurate and rapid stem-loop reverse transcriptional real-time PCR (RT-PCR) method to quantify human let-7a miRNA in gastric cancer.

Methods: According to the sequence of let-7a miRNA, the stem-loop reverse transcriptional primer, the primers and quantitative MGB probes of real-time PCR were designed and synthesized. The dynamic range and the sensitivity of quantitative reverse transcriptional real-time PCR were determined. The levels of let-7a miRNA were examined in 32 gastric carcinoma samples by stem-loop RT-PCR method.

Results: The dynamic range and sensitivity of the let-7a miRNA quantification scheme were evaluated, the result showed the assay could precisely detect 10 copies of mature let-7a miRNA in as few as 0.05 ng of total RNA of gastric mucosa. The results of specificity analysis showed no fluorescence signal occurred even though 50 ng of human genomic DNA was added to the reverse transcription (RT) reaction. The expression level of let-7a miRNA in gastric tumor tissues was significantly lower compared to normal tissues in 14 samples from 32 patients.

Conclusion: The stem-loop RT-PCR is a reliable method to detect let-7a miRNA which may play an important role in the development of gastric carcinoma.

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Figures

Figure 1
Figure 1
Sensitivity and dynamic range of let-7a miRNA assay. A: Amplification curve of the synthesized let-7a miRNA, the well-behaved assay exhibited high sensitivity and broad dynamic range between the signal of 10 copies and 108 copies input template and the no-template-control background; B: Standard curve of the let-7a miRNA (r2 = 0.996, slope = -2.92).
Figure 2
Figure 2
Total RNA sensitivity and dynamic range of let-7a miRNA assay. A: Amplification plot of total RNA from gastric mucosa at six orders of magnitude. The total RNA input ranged from 50 pg to 5 μg in per stem-loop RT-PCR reaction; B: Correlation of total RNA input with the threshold of cycle (Ct) values for let-7a miRNA assays.
Figure 3
Figure 3
Expression of let-7a miRNA in gastric cancer. Difference in let-7a miRNA expression between in tumor tissues (T) and the adjacent non-tumor tissues (N). The patient numbers correspond to those in Table 2. Compared to normal tissues, let-7a miRNAs expression in tumor tissue from 14 patients was significantly down-regulated.
See this image and copyright information in PMC

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