Maximizing RNA yield from archival renal tumors and optimizing gene expression analysis.
Maximizing RNA yield from archival renal tumors and optimizing gene expression analysis.
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DOI：
10.1177/1087057109355059
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发表时间：
2010-01
影响因子：
--
通讯作者：
Kim HL
中科院分区：
文献类型：
--
作者：
Glenn ST;Head KL;Teh BT;Gross KW;Kim HL
福尔马林固定石蜡包埋组织广泛用于TaqMan PCR基因表达分析。比较了从石蜡包埋肾肿瘤组织中提取RNA的5种方法，包括4种商用试剂盒。Epicentre的MasterPure试剂盒的RNA产量最高。但是，Epicenter的试剂盒与Invitrogen的Trizol方法的RNA产率差异不显著。使用前3种RNA分离方法，对制造商的方案进行修改，包括过夜蛋白酶K消化。隔夜蛋白质消化导致RNA产量显著增加。为了通过反转录提高cDNA的生成，我们将随机寡核苷酸引物与针对目标基因的基因特异性引物进行了比较。利用基因特异性引物进行反转录，可显著提高TaqMan PCR检测到的cDNA数量。因此，通过使用Masterpure RNA分离试剂盒进行修饰，包括过夜蛋白酶K酶切和逆转录过程中的基因特异性引物，可以优化使用TaqMan qPCR对福尔马林固定石蜡包埋组织的表达谱分析。
Formalin-fixed paraffin-embedded tissues are widely available for gene expression analysis using TaqMan PCR. Five methods, including 4 commercial kits, for recovering RNA from paraffin embedded renal tumor tissue were compared. The MasterPure kit from Epicentre produced the highest RNA yield. However, the difference in RNA yield between the kit from Epicenter and Invitrogen’s Trizol method was not significant. Using the top 3 RNA isolation methods, the manufacturers’ protocols were modified to include an overnight Proteinase K digestion. Overnight protein digestion resulted in significant increase in RNA yield. To improve cDNA production by reverse transcription, random oligonucleotide primers were compared to gene-specific primers targeting the genes of interest. Reverse transcription using gene-specific primers significantly increased the quantity of cDNA detectable by TaqMan PCR. Therefore, expression profiling of formalin-fixed paraffin-embedded tissue using TaqMan qPCR can be optimized by using the Masterpure RNA isolation kit modified to include an overnight proteinase K digestion and gene-specific primers during the reverse transcription.
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影响因子：
2.7
作者：
Glenn, Sean T.;Jones, Craig A.;Kim, Hyung L.
通讯作者：
Kim, Hyung L.
影响因子：
4.1
作者：
Doleshal, Martina;Magotra, Amber A.;Szafranska, Anna E.
通讯作者：
Szafranska, Anna E.
影响因子：
2.7
作者：
Abramovitz, Mark;Ordanic-Kodani, Maja;Leyland-Jones, Brian
通讯作者：
Leyland-Jones, Brian
影响因子：
--
作者：
Chung, Joon-Yong;Braunschweig, Till;Hewitt, Stephen M.
通讯作者：
Hewitt, Stephen M.
影响因子：
6
作者：
Cronin, M;Pho, M;Baker, JB
通讯作者：
Baker, JB