Successful downstream application of the Paxgene Blood RNA system from small blood samples in paediatric patients for quantitative PCR analysis.
Successful downstream application of the Paxgene Blood RNA system from small blood samples in paediatric patients for quantitative PCR analysis.
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DOI：
10.1186/1471-2172-8-20
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发表时间：
2007-09-12
期刊：
影响因子：
3
通讯作者：
Day P
中科院分区：
文献类型：
--
作者：
Carrol ED;Salway F;Pepper SD;Saunders E;Mankhambo LA;Ollier WE;Hart CA;Day P
基因表达研究的挑战是可靠地量化转录水平，但这受到许多因素的阻碍，包括样品的可用性，处理和储存。PAXgene™血液RNA系统在塑料真空管中包含稳定添加剂，但需要2.5 mL血液，这使得儿科使用的常规实施不切实际。本研究的目的是修改PAXgene™血液RNA系统试剂盒方案，以应用于小的患病儿童，而不影响RNA的完整性，并随后进行ICAM和白细胞介素-6基因表达的定量分析。将0.86 mL PAXgene™试剂的等分试样放入微管中，并加入0.3 mL全血以保持与PAXgene™真空管系统中相同的推荐比例。使用Agilent BioAnalyser 2100和内部TaqMan™测定法评估RNA质量，所述测定法通过测定3'与5'比率来测量GAPDH转录物完整性。对另外一组7个管家基因进行qPCR分析。使用GeNORM算法鉴定了三个参考基因（HPRT 1、YWHAZ和GAPDH），随后将其用于标准化靶基因表达水平。ICAM-1和IL-6基因表达测定87马拉维儿童侵袭性肺炎球菌疾病。总RNA产量在1，114和2，950 ng之间，BioAnalyser 2100显示出可辨别的18 s和28 s条带。获得的7个管家基因的循环阈值在15和30之间，并表现出良好的一致性。与存活者相比，非存活者中的中位相对ICAM和IL-6基因表达显著降低（ICAM：3.56 vs 4.41，p = 0.04，IL-6：2.16 vs 6.73，p = 0.02）。我们已经成功地修改了PAXgene™血液采集系统用于幼儿，并证明了RNA完整性的保存和成功的定量实时PCR分析。
The challenge of gene expression studies is to reliably quantify levels of transcripts, but this is hindered by a number of factors including sample availability, handling and storage. The PAXgene™ Blood RNA System includes a stabilizing additive in a plastic evacuated tube, but requires 2.5 mL blood, which makes routine implementation impractical for paediatric use. The aim of this study was to modify the PAXgene™ Blood RNA System kit protocol for application to small, sick chidren, without compromising RNA integrity, and subsequently to perform quantitative analysis of ICAM and interleukin-6 gene expression. Aliquots of 0.86 mL PAXgene™ reagent were put into microtubes and 0.3 mL whole blood added to maintain the same recommended proportions as in the PAXgene™ evacuated tube system. RNA quality was assessed using the Agilent BioAnalyser 2100 and an in-house TaqMan™ assay which measures GAPDH transcript integrity by determining 3' to 5' ratios. qPCR analysis was performed on an additional panel of 7 housekeeping genes. Three reference genes (HPRT1, YWHAZ and GAPDH) were identified using the GeNORM algorithm, which were subsequently used to normalising target gene expression levels. ICAM-1 and IL-6 gene expression were measured in 87 Malawian children with invasive pneumococcal disease. Total RNA yield was between 1,114 and 2,950 ng and the BioAnalyser 2100 demonstrated discernible 18s and 28s bands. The cycle threshold values obtained for the seven housekeeping genes were between 15 and 30 and showed good consistency. Median relative ICAM and IL-6 gene expression were significantly reduced in non-survivors compared to survivors (ICAM: 3.56 vs 4.41, p = 0.04, and IL-6: 2.16 vs 6.73, p = 0.02). We have successfully modified the PAXgene™ blood collection system for use in small children and demonstrated preservation of RNA integrity and successful quantitative real-time PCR analysis.
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影响因子：
3.3
作者：
RIECKMANN, P;NUNKE, K;FELGENHAUER, K
通讯作者：
FELGENHAUER, K
DOI：
10.4049/jimmunol.177.9.6480
发表时间：
2006-11-01
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
--
作者：
Marriott HM;Hellewell PG;Cross SS;Ince PG;Whyte MK;Dockrell DH
通讯作者：
Dockrell DH
影响因子：
3.1
作者：
Thornton, J;McDaniel, LS
通讯作者：
McDaniel, LS
影响因子：
6.4
作者：
Winkler, F;Koedel, U;Pfister, HW
通讯作者：
Pfister, HW
影响因子：
9.3
作者：
Zheng, Z;Luo, YL;McMaster, GK
通讯作者：
McMaster, GK