Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA
Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA
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DOI：
10.2144/000112153
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发表时间：
2006-05-01
期刊：
影响因子：
2.7
通讯作者：
Dufva, Martin
中科院分区：
文献类型：
--
作者：
Stangegaard, Michael;Dufva, Inge Hagh;Dufva, Martin
RNA的逆转录是通过实时PCR或微阵列方法进行基因表达分析的一种极有价值的方法。对不同长度的随机引物在引发逆转录反应的效率方面进行了比较。结果表明，以poly(A) RNA或信使RNA的扩增形式（aRNA）为模板时，15个核苷酸长的随机寡核苷酸（十五聚体）产生的cDNA始终至少是随机六聚体的2倍。与随机六聚体产生的cDNA相比，使用十五聚体产生的cDNA在大小分布或掺入标记的量方面没有差异。使用随机十五聚体引发效率的提高使得>80%的模板aRNA发生逆转录，而随机六聚体仅诱导40%的模板aRNA发生逆转录。这表明使用随机十五聚体比随机六聚体对转录组有更好的覆盖。在全转录组DNA微阵列实验中，以相同量的aRNA作为起始材料，随机十五聚体引发的反应检测到的基因比随机六聚体引发的反应多11倍。结果表明，在对poly(A) RNA和扩增RNA的逆转录反应中，随机十五聚体可以替代随机六聚体，从而产生更高的cDNA产量和质量。
Reverse transcription of RNA is an invaluable method for gene expression analysis by real-time PCR or microarray methods. Random primers of varying lengths were compared with respect to their efficiency of priming reverse transcription reactions. The results showed that l5-nucleotide-long random oligonucleotides (pentadecamers) consistently, yielded at least 2 fold as much cDNA as did random hexamers using either-poly(A) RNA or an amplified version of messenger RNA (aRNA) as a template. The cDNA generated using pentadecamers did not differ in size distribution or the amount of incorporated label compared with cDNA generated with random hexamers. The increased efficiency of priming using random pentadecamers resulted in reverse transcription of > 80% of the template aRNA, while random hexamers induced reverse transcription of only 40% of the template aRNA. This suggests a better coverage of the transcriptome when using random pentadecamers over random hexamers. Using the same amount of aRNA as starting material, random pentadecamer-printed reactions resulted in 11-fold more genes being detected in whole transcriptome DNA microarray experiments than random hexamer-primed reactions. The results indicate that random pentadecamers can replace random hexamers in reverse transcription reactions on both poly(A) RNA and amplified RNA, resulting in higher cDNA yields and quality.