Results Purified RNA samples were quantitated by absorbance using a NanoDrop® ND-1000 instrument. The average RNA yield for peripheral blood (PB) was 216ng/µl and for bone marrow (BM) was 415ng/µl in a 40μl volume. Purity was determined by measuring absorbance at 260 and 280nm and calculating the A260 /280 ratio. RNA Integrity An Agilent 2100 Bioanalyzer Instrument was used to determine RNA integrity numbers (RIN) as a measure of RNA quality, with numbers >8 representing good-quality RNA. Representative PB RNA samples (n=23) were analyzed using the Agilent RNA 6000 Nano Kit. Samples had mean RIN values >9.3 with a range of 9–9.8, indicating high-quality RNA. qPCR Analysis Amplification of an endogenous control gene such as ABL is used as a reference to normalize target transcript levels and identify poor quality samples. ABL is the most common reference gene used due to its wide expression level and stability across different cell types. The ability to amplify at least 10,000 ABL molecules per reaction provides, in our experience, a safety measure against poor sample quality, avoiding incorrect normalization in quantitative assay and also false-negative results. By assessing the absolute value of the ABL housekeeping gene we estimated whether quantification results could be affected by the purification method. As shown in Figure 1, all samples revealed good RNA quality, with excellent performance in qPCR and nearly all of the samples (above 95%) showed an ABL copy number above 10,000, thus resulting in samples that are suitable for most qualitative downstream applications. 1 × 106 1 × 105 1 × 104 1 × 103 Total Bone Marrow Peripheral Blood Read the Complete Technical Article » enews | Spring 2016 Figure 1. Sample quality distribution measured by ABL copy number using two different qPCR systems. Total RNA extracted from cells was used as a template for cDNA transcription. After first-strand synthesis the reaction was treated with E. coli RNase H to remove the RNA template from the cDNA:RNA hybrid molecule. Samples (n = 450; 205 BM and 245 PB) were screened on the iCycler IQ apparatus and ABL real-time qPCR assays were performed using 2X IQ™ Supermix (Bio-Rad Cat.# 170-8862) and ABL standards (Qiagen Cat.# 674691). In addition, some samples (n=251; 138 BM and 113 PB) underwent real-time qPCR analysis for ABL detection on Applied Biosystems platform using ABL mix from ProfileQuant® kit (Qiagen Cat.# 676923) or from Alert kit (ELITechGroup, Cat.# RTSG07-210). ABL Copy Number 11918MA Bio-Rad ABI 7300 Bio-Rad ABI 7300 Bio-Rad ABI 7300 Pagina 12
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