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Microarrays: Human Disease Detection and Monitoring
Published in Attila Lorincz, Nucleic Acid Testing for Human Disease, 2016
Janet A. Warrington, Thomas B. Broudy
Acceptance of microarray data for use in clinical applications requires standardization of technical performance assessment methods across the industry. To this end, over 70 scientists from public, private, and academic institutions have formed the External RNA Controls Consortium (ERCC, http://www.cstl.nist.gov/biotech/Cell&TissueMeasurements/GeneExpression/ERCC.htm) with the goal of developing external RNA spike-in controls and recommended protocols for sample control on gene expression technologies such as microarrays and quantitative real-time reverse transcriptase PCR.89 The controls will consist of 100 well characterized clones comprised of randomly generated unique sequences as determined by sequence comparison to mouse, rat, human, Drosophila, Escherichia coli, mosquito sequence databases, and Bacillus subtillus and Arabidopsis thaliana clones. Control sequences will be tested in the second half of 2006 by multiple labs participating in the ERCC including all major microarray manufacturers, the National Institute of Standards and Technology, the Food and Drug Administration, and the National Cancer Institute. All data will be made public.90
Comprehensive profiling of Small RNAs in human embryo-conditioned culture media by improved sequencing and quantitative PCR methods
Published in Systems Biology in Reproductive Medicine, 2020
Stewart J. Russell, Karen Menezes, Hanna Balakier, Clifford Librach
Reconstituted ECCM RNA was spiked with custom RNA spike-in set of 10 oligos, designed similar to (Lutzmayer et al. 2017). For the purposes of this study, the spike-in sequences provided confirmation of successful library prep from both methylated and non-methylated sncRNAs. Spike-in dilution and sequencing results details are contained in Supplemental Figure 2 and Table 4. SncRNA libraries were prepared using NEXTFLEX® Small RNA-Seq Kit v3 (Bioo Scientific). Small RNAs were ligated with adapters having randomized ends and reverse transcription was performed. The resulting cDNA was barcoded with unique kit barcodes and amplified. Gel free size selection of libraries was performed using Blue pippin. Traces of amplified and size selected libraries were assessed using Bioanalyzer 2100 (Agilent technologies). The libraries were denatured and diluted according to the manufacturer’s protocols using NextSeq 500/550 High output kit v2 (75 cycles). Sequencing was carried out on NextSeq 550 instrument (Illumina, San Diego, California)
Circulating miRNA-33: a potential biomarker in patients with coronary artery disease
Published in Biomarkers, 2019
Lakshmi Lavanya Reddy, Swarup A.V. Shah, Chandrashekhar K. Ponde, Rajesh M. Rajani, Tester F. Ashavaid
For qRT-PCR, 5 µL of the cDNA product was used as template in 20 µL reaction containing ExiLENT SYBR® Green PCR reaction buffer, enzyme mix and synthetic spike-in. The RNA spike-in kit Exiqon A/S was used according to the manufacturer’s instructions. qRT-PCR was performed for all five miRNAs along with housekeeping or reference gene, miR-451 with 7500 FAST real-time PCR system (Applied Biosystems, Foster, CA, US) at 95 °C for 10 min followed by 45 amplification cycles at 95 °C for 10 s and 60 °C for 1 min. miR-451 was used as an internal standard because it was detectable in all samples and its normalized intensities were not significantly different between CAD cases and controls plasma samples (p > 0.05). Triplicate measurements were obtained for each sample. Data were analysed with SDS Relative Quantitation Software version 1.4 with the automatic Ct setting for assigning baseline and threshold for Ct determination. The relative expression level of each individual miRNA levels were calculated using the 2–ΔΔCt method which were normalized with the housekeeping or reference miR-451, (ΔCt = CtmiR of sample x – Ct451 of sample x). The Ct values from qRT-PCR assays between 15 and 40 were considered to be expressed.
High-throughput tool to discriminate effects of NMs (Cu-NPs, Cu-nanowires, CuNO3, and Cu salt aged): transcriptomics in Enchytraeus crypticus
Published in Nanotoxicology, 2018
Susana I. L. Gomes, Carlos P. Roca, Natália Pegoraro, Tito Trindade, Janeck J. Scott-Fordsmand, Mónica J. B. Amorim
RNA was extracted from each replicate containing a pool of 20 animals. Three biological replicates per test treatment (including controls) were used. Total RNA was extracted using SV Total RNA Isolation System (Promega, Madison, WI). The quantity and purity of the isolated RNA were measured spectrophotometrically with a nanodrop (NanoDrop ND-1000 Spectrophotometer, Wilmington, DE) and its quality was checked on a denaturing formaldehyde agarose gel electrophoresis. A single-color design was used. In brief, 500 ng of total RNA was amplified and labeled with Agilent Low Input Quick Amp Labelling Kit (Agilent Technologies, Palo Alto, CA). Positive controls were added with the Agilent one-color RNA Spike-In Kit (Agilent Technologies, Palo Alto, CA). Purification of the amplified and labeled cRNA was performed with the RNeasy columns (Qiagen, Valencia, CA).